PROGRAMME and ABSTRACTS

Transcription

Organizer:
Polish Neuroscience Society (PNS)
Co-organizers:
Federation of European Neuroscience Societies (FENS), Committee of Neurobiology of Polish
Academy of Sciences (CN PAS), Committee of Neurological Sciences of Polish Academy of
Sciences (CNS PAS)
Partners:
Nencki Institute of Experimental Biology PAS, Mossakowski Medical Research Centre PAS,
Institute of Pharmacology PAS, International Institute of Molecular and Cell Biology
Chairperson:
Jolanta Skangiel-Kramska - President of the Polish Neuroscience Society
Scientific Program Committee:
Chairperson:
Vice-Chairpersons:
Jolanta Skangiel-Kramska
Irena Nalepa, Elżbieta Pyza
Members:
Julita Czarkowska-Bauch, Henryk Majczyński, Adam Płaźnik, Małgorzata Skup, Teresa
Zalewska (PNS), Helmut Kettenmann, Eckart Gundelfinger (FENS)
Local Organizing Committee:
Chairperson:
Vice-Chairperson
Małgorzata Skup
Katarzyna Łukasiuk
Secretary:
Treasurer:
Members:
Teresa Zalewska
Henryk Majczyński
Paweł Boguszewski (webpage, poster sessions, audiovisual equipment), Ruzanna Djavadjan
(book exhibition), Robert Filipkowski (sponsors and exhibitors, exhibition prospectus, Lem
“The futurological Congress” project), Katarzyna Kalita (exhibitors), Ksenia Meyza (animation
of the events for stipend holders), Urszula Sławińska (media), Tomasz Werka (graphic design
and announcements layouts), Marek Wypych (animation of the events for stipend holders)
FENS Office Berlin:
Meino Gibson
Organizers of Brain Awareness Events:
History of Neuroscience in Poland – exhibition (In Polish and in English): Andrzej Wróbel
EDAB Open Lecture for Public (In Polish): Jolanta Skangiel-Kramska, Teresa Zalewska
Strategic sponsor:
Kawa.ska & Leica
Sponsors:
Ministry of Science and Higher Education of Poland
International Brain Research Organization (IBRO)
European Dana Alliance for the Brain (EDAB)
European Brain and Behaviour Society (EBBS)
University of Warsaw
The British Neuroscience Association (BNA)
German Neuroscience Society (GNS)
Olympus
Millipore
WPI
Professional Partner:
CED
ABE
Jackson Immunoresearch
Applied Biosystems
ZEISS
Bioseb
InLab
Enpol
ProVita
Nobell Congressing: Norbert Karczmarczyk, Katarzyna Wejsis, Bartosz Czepułkowski (webpage)
Programme 265
NINTH INTERNATIONAL CONGRESS
OF THE POLISH NEUROSCIENCE SOCIETY
First FENS Featured Regional Meeting
PROGRAMME
Wednesday, September 9
12.00 – 18.00
Registration
14.00 – 16.00
General Assembly of the Polish Neuroscience Society
location: Old Library, Warsaw University, Krakowskie Przedmieście 26/28
17.00 – 18.30
Opening Session
location: Old Library, Warsaw University, Krakowskie Przedmieście 26/28
Welcome Addresses (Jolanta Skangiel-Kramska, President of Polish Neuroscience Society
and guests)
Opening Lecture
Sponsored by: Kawa.ska
Oswald Steward (Reeve-Irvine Research Center, University of California at Irvine, CA, USA)
Targeting of mRNA to postsynaptic sites on neuronal dendrites
Introduced by: Malgorzata Skup
19.30
08.30 – 09.20
Concert of Polish Ancient Music (Ars Nova Ensamble) and Get-Together Reception
location: Central Agricultural Library, Krakowskie Przedmieście 66
Thursday, September 10
Plenary Lecture
Pasko Rakic (Yale University School of Medicine, New Haven, CT, USA)
Making map of the mind: Molecular mechanisms of neuronal migration
Introduced by: Malgorzata Kossut
09.20 – 09.40
Young Investigator Lecture (PNS award)
Ksenia Meyza (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
Electrophysiological correlates of diverse emotional reactivity of Roman High - (RHA/Verh)
and Roman Low Avoidance (RLA/Verh) rats
Introduced by: Elzbieta Pyza
09.40 – 10.00
Coffee break
10.00 – 11.30
SYMPOSIA
Symposium I: Learning-Dependent Brain Plasticity: Systems View
Organizer and Chair: Grzegorz Hess (Jagiellonian University and Institute of Pharmacology
PAS, Krakow, Poland)
Carl Petersen (Mind Brain Institute, Lausanne, Switzerland)
Synaptic mechanisms of tactile sensory properties
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Wioletta Waleszczyk (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
Reorganization of the visual cortex following monocular circumscribed retinal lesions: Update on
Hebbian learning rules
Mathew Diamond (SISSA, Trieste, Italy)
What is stored in the hippocampus during a tactile categorization task?
Malgorzata Kossut (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
The learning brain – modulation of activation pattern
Symposium II: Using Neuroimaging to Understand Human Brain Function
Organizer and Chair: Anna Grabowska, Anna Nowicka (Nencki Institute of Experimental
Biology PAS, Warszawa, Poland)
Piotr Bogorodzki (Institute of Electronics, Warszawa, Poland)
Inside BOLD – why neuronal activity modulates MR signal?
Greg Kroliczak (University of Oregon, Eugene, OR, USA)
Neural bases of praxis and target-directed actions
Stefan Heim (RWTH Aachen University, Jülich, Germany)
The neural correlates of language processing and its disorders over the life span
Artur Marchewka (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
Neural correlates of false recognition
9.40 – 11.40
Workshop: Quantitative Analysis of Protein–Protein Interactions
(Support contributed by Kawa.ska and Deutsche Forschungsgemeinschaft)
Organizer and chair: Jakub Wlodarczyk (Nencki Institute of Experimental Biology PAS Warszawa,
Poland)
Erwin Neher (Max-Plank-Institute for Biophysical Chemistry, Göttingen, Germany)
Non-negative matrix factorization: A novel method to determine reference spectra of fluorescent dyes
in a cellular environment
Diethelm Richter (University of Göttingen, Göttingen, Germany)
Quantitative measurements of cAMP concentration with a new Epac Based FRET-Sensor
Evgeni Ponimaskin (Medical School Hanover, Hanover, Germany)
Stimulation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors
Leszek Kaczmarek, Jakub Wlodarczyk (Nencki Institute of Experimental Biology PAS, Warszawa,
Poland)
Lux-FRET method for quantitative analysis of FRET signal
Grzegorz Wilczynski (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
High-resolution light-microscopic studies on synaptic extracellular proteolysis
11.30 – 12.00
Coffee break
12.00 –12.50
Plenary Lecture
Jerzy Lazarewicz (Mossakowski Medical Research Centre PAS, Warszawa, Poland)
Alternative methods of neuroprotection in brain ischemia
Introduced by: Krystyna Domanska-Janik
12.50 – 14.30
Lunch
14.30 – 16.30
POSTER SESSION I
location: Warsaw Academy of Fine Arts, Krakowskie Przedmieście 5
Programme 267
16.30 – 18. 00
SYMPOSIA
Symposium III: Learning-Dependent Brain plasticity: Synaptic View
Organizer and Chair: Malgorzata Kossut (Nencki Institute of Experimental Biology PAS,
Warszawa, Poland)
Denise Manahan-Vaughan (Ruhr University, Bochum, Germany)
Regional and functional specificity of hippocampal synaptic plasticity in spatial memory
formation
Krzysztof Tokarski (Institute of Pharmacology PAS, Kraków, Poland)
Sensory learning enhances GABAergic synaptic transmission in the barrel cortex of the mouse
Jerzy Mozrzymas (Wroclaw Medical University, Wrocław, Poland)
Impact of sensory learning on GABAergic tonic inhibition in mouse barrel cortex
Alison Barth (Carnegie-Mellon University, Pittsburgh, PA, USA)
Synaptic metaplasticity in somatosensory cortex
Symposium IV: Mechanisms, Functions and Modulation of Central Nervous System
Inflammation
Organizer and Chair: Bozena Kaminska (Nencki Institute of Experimental Biology PAS,
Warszawa, Poland)
Wolfgang J. Streit (University of Florida College of Medicine, Gainesville, Florida, USA)
Microglial senescence and degeneration in the pathogenesis of neurodegenerative disease
Anna Czlonkowska (Institute of Psychiatry and Neurology, Warszawa, Poland)
Manipulating inflammation and gliosis in neurologic diseases – a clinical perspective
Manuel B. Graeber (Imperial College, London, UK)
Glia and inflammation in the Parkinsonian substantia nigra
Barbara Przewlocka (Institute of Pharmacology PAS, Kraków, Poland)
Modulation of inflammation and neuropathic pain
Symposium V: Stress-Response Systems in the Pathogenesis and Prognosis of Cancer,
Inflammation and Autoimmune Diseases
Organizer and Chair: Marta Kubera (Institute of Pharmacology PAS, Kraków, Poland)
Eadaoin Griffin (National University of Ireland, Maynooth, Ireland)
Psychological stress compromises the innate immune system: Implications for disease susceptibility
Danuta Lewandowska (University of Gdansk, Gdańsk, Poland)
Stress, emotion, and natural killer cell antitumor activity
Lisa Goehler (University of Virginia, Charlottesville, Virginia, VA, USA)
Role of stress on immunity and cancer development
Craig Stockmeier (University of Mississippi Medical Center, Jackson, MS, USA)
Cell pathology and plasticity in the hippocampus in major depressive disorder
18.00 – 18.10
Coffee break
18.10 – 18.30
Young Investigator Lecture (FENS stipend holder award)
Christian Wozny (MRC Laboratory of Molecular Biology, Cambridge, UK)
Looking at the top of the cortex: Neural connectivity in the superficial layers
of the neocortex
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18.30 – 19.20
Plenary Lecture
Grazyna Rajkowska (University of Mississippi Medical Center, Center for Psychiatric
Neuroscience, Jackson, MS, USA)
Glia, GABA and Glutamate: New key players in the pathology of depression
Introduced by: Irena Nalepa
19.30 – 20.15
Foundation for Polish Science presentation for FENS stipend holders
20.30
Welcome reception (sponsored by FENS)
location: Le Jardin Restaurant, Zamojski Palace, Foksal 2
Friday, September 11
08.30 – 09.20
Plenary lecture
Jerzy Samochowiec (Pomeranian Medical University, Szczecin, Poland)
Alcoholism: From neurobiology to new treatment approach
09.20 – 09.40
Lukasz Swiech (Institute of Molecular and Cell Biology, Warszawa, Poland)
TIPs for dendritogenesis: CLIP-170 in dendritic arbor development
Introduced by: Adam Plaznik
09.40 – 10.00
Coffee break
10.00 – 11.30
SYMPOSIA
Symposium VI: Genes and Behavior
Organizer and Chair: Artur H. Swiergiel (Institute of Biology, University of Gdansk, Gdańsk,
Poland)
Grzegorz R. Juszczak (Institute of Genetics and Animal Breeding PAS, Jastrzebiec, Poland)
It is said “almost makes a difference”: Are different strains of rodents the same or almost the same?
Dora Zelena (Group of Neuropsychopharmacology HAS and Semmelweis University, Budapest,
Hungary)
Stress and adaptation in Brattleboro rats lacking arginine-vasopressin
Aet Alttoa (Department of Psychology, University of Tartu, Estonia)
Measurement of persistently expressed behavioural traits in outbred rats: Underlying gene
expression patterns
Symposium VII: Stem Cells Based Therapeutic Approaches
(Support contributed by Olympus Polska)
Organizer and Chair: B. Lukomska, K. Domanska-Janik (Mossakowski Medical Research Centre
PAS, Warszawa, Poland)
Marcin Jurga (Institute Novus Sanguis, Parc Technologique, St Priest, Lyon, France)
Artificial neural tissue from cord blood for tissue replacement therapy central nervous system
Kyung-Sun Kang (Adult Stem Cell Research Center, Seoul National University, Seoul, Korea)
Multi-functional therapeutic potentials of human umbilical cord blood-derived stem cell
in neurodegenerative diseases
Klaus Reymann (Leibnitz Institute for Neurobiology, Magdeburg, Germany)
Stem cell transplantation in rodent stroke models
Programme 269
Symposium VIII: Cholinergic System in Neurodegereneration
Organizer and Chair: Andrzej Szutowicz (Medical University of Gdansk, Gdańsk, Poland)
Natalia Nalivaeva (University of Leeds, Leeds, UK)
Amyloid precursor protein and cholinesterase in brain function and neurodegeneration
Reinhardt Schliebs (University of Leipzig, Leipzig, Germany)
Cholinotoxicity of β-amyloid
Anthony Turner (University of Leeds, Leeds, UK)
Proteolytic cascades in the brain and their cholinergic regulation: Role in neurodegeneration
Andrzej Szutowicz (Medical University of Gdansk, Gdańsk, Poland)
Phenotype and energy-dependent cholinotoxicity
11.30 – 11.45
Coffee break
11.45 – 11.55
FENS address
Helmut Kettenmann – President of FENS
12.00 – 12.50
Plenary lecture
Morgan Sheng (Genentech Inc., South San Francisco, CA, USA)
Regulation of synaptic function by modification of postsynaptic density proteins
Introduced by: Jacek Jaworski
12.50 – 14.30
Lunch
14.30 – 16.30
POSTER SESSION II
location: Warsaw Academy of Fine Arts, Krakowskie Przedmiescie 5
16.30 – 18.00
SYMPOSIA
Symposium IX: Protein Toxicity and Molecular Mechanisms of Neurodegenerative Diseases
Organizer and Chair: Robert Strosznajder (Mossakowski Medical Research Centre PAS,
Warszawa, Poland)
Shun-ichi Nakamura (Kobe University, Graduate School of Medicine, Kobe, Japan)
Sphingosine-1-phosphate in physiology and pathology in the brain
Michal Toborek (University of Kentucky, Lexington, KY, USA)
Role of caveolin-1 protein in signaling mechanism and in endothelial cell function and dysfunction
Agata Adamczyk (Mossakowski Medical Research Centre PAS, Warszawa, Poland)
Alpha synuclein and its neurotoxic fragment NAC in neurodegeneration
Martin Sadowski (New York University School of Medicine, New York, NY, USA)
Therapeutic approaches targeting Aβ cascade in Alzheimer’s disease
Symposium X: Stroke-Induced Brain Reorganization
Organizer and Chair: Jolanta Skangiel-Kramska (Nencki Institute of Experimental Biology
PAS, Warszawa, Poland)
Mathias Hoehn (Max-Planck Institute for Neurological Research, Köln, Germany)
Exploring the chances of stem cell based regeneration after stroke by MRI
Dorota Nowicka (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
How phothotrombotic stroke affects extracellular matrix?
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Małgorzata Kossut (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
fMRI study of recovery from unilateral neglect
Otto Witte (Friedrich Schiller University, Jena, Germany)
Brain plasticity following stroke: Possible targets for therapeutic intervention
Symposium XI: Glutamatergic receptors as a target for future psychotropic drugs
action
Organizer and Chair: Andrzej Pilc (Institute of Pharmacology PAS, Kraków, Poland)
Barbara Wroblewska (Georgetown University, Washington DC, USA)
Role of group II metabotropic glutamate receptors in schizophrenia models
Wojciech Danysz (Merz Pharmaceuticals, Frankfurt am Main, Germany)
Selected aspects of Group I mGlu receptor negative and positive modulators
Krystyna Ossowska (Institute of Pharmacology PAS, Kraków, Poland)
Involvement of mGlu receptors in ethiopathology of Parkinson’s disease
Andrzej Pilc (Institute of Pharmacology PAS, Kraków, Poland)
Metabotropic glutamate receptor ligands as possible targets for treatment of anxiety and
depression
18.00 – 18.15
Coffee break
18.15 – 18.45
EJN Lecture (EJN award)
Ji H. Ko (McGill University, Montréal, Canada)
Theta burst stimulation of dorsolateral prefrontal cortex reveals hemispheric
asymmetry in striatal dopamine release during set-shifting task in human
Chair: Jean-Marc Fritschy (Institute of Pharmacology and Toxicology, University of Zurich,
Zürich, Switzerland)
18.45 – 19.35
Plenary Lecture
Annamaria Vezzani (“Mario Negri” Institute for Pharmacological Research, Milano, Italy)
Cytokine-mediated cross-talk between glia and neurons: Neuropathological
implications for epilepsy
Introduced by: Katarzyna Lukasiuk
20.30
FENS Dinner (by invitation only)
Location: “Belvedere” Restaurant, Royal Łazienki Park
Saturday, September 12
08.30 – 09.20
Plenary lecture
Bozena Kaminska (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
Exploring and manipulating function of microglia in brain pathologies
Introduced by: Ryszard Przewlocki
09.20 – 09.40
Young Investigator Lecture (FENS stipends holder award)
Piotr Walczak (Johns Hopkins University School of Medicine, Baltimore, MD, USA)
Guiding cell-based therapy for neurological diseases with noninvasive cellular imaging
09.40 – 10.00
Coffee break
Programme 271
10.00 – 11.30
SYMPOSIA
Symposium XII: Interaction Between Antidepressant Drugs and Immunoendocrine Systems
Organizer and Chair: Boguslawa Budziszewska, Wladyslaw Lason (Institute of Pharmacology
PAS, Kraków, Poland)
Peter Gass (Central Institute of Mental Health, Mannheim, Germany)
Mice with altered glucocorticoid receptor expression as a model for depression
Ewa Obuchowicz (Medical University of Silesia, Katowice, Poland)
Effects of antidepressant drugs on cytokines in experimental study
Francesco Roselli (Max-Planck Institute of Psychiatry, München, Germany)
Neuroplasticity in depression
Marta Kubera (Institute of Pharmacology PAS, Kraków, Poland)
Immunoendocrine changes in animal model of depression
Symposium XIII: Motor Units and Motoneurons
Organizer and Chair: Jan Celichowski (University School of Physical Education, Poznań, Poland)
Philip Gardiner (University of Mannitoba, Winnipeg, Canada)
Physiological adaptations in the rhytmic firing properties of alpha-motoneurons to changes in
physical activity and aging
Claudio Orizio (University of Brescia, Brescia, Italy)
Motor units activity in EMG, MMG and force ripple: Three pictures for one subject
Piotr Krutki (University School of Physical Education, Poznań, Poland)
Contractions of motor units evoked by stimulation with pulses at variable interpulse intervals
Jan Celichowski (University School of Physical Education, Poznań, Poland)
Variability and plasticity of motor unit contractile properties
Symposium XIV: Advanced Methods of Brain Signal Analysis
Organizer and Chair: Katarzyna Blinowska (Warsaw University, Faculty of Physics, Warszawa,
Poland)
Fabrizio De Vico Fallani (University of Rome La Sapienza, Rome, Italy)
The use of high resolution EEG methods for brain computer interface applications
Daniel Wojcik (Nencki Institute of Experimental Biology PAS, Warszawa, Poland)
Beyond the PSTH: Point process modeling of spike trains
Stilian Kalitzyn (Epilepsy Institute of The Netherlands – SEIN, Heemstede, The Netherlands)
Perturbing the epileptic brain, evoked responses and beyond
Maciej Kaminski (Faculty of Physics, Warsaw University, Warszawa, Poland)
Evaluation of dynamical information transfer in brain
11.30 – 12.00
Coffee break
12.00 – 12.30
Luiza Wojcik (Mossakowski Medical Research Centre PAS, Warszawa, Poland)
Relationship between activity of matrix metalloproteinase -2 and -9 (MMP-2, MMP-9) and
ischemia-induced neurogenesis in adult gerbil hippocampus
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12.30 – 13.20
Plenary lecture
(Support contributed by EBBS)
Mark Tuszynski (University of California, Los Angeles, CA, USA)
Growth factor gene delivery for Alzheimer’s disease: From animal models to clinical trials
Introduced by: Julita Czarkowska-Bauch
13.30
Closing ceremony
BRAIN AWARENESS EVENTS
Saturday, September 12th
16.00 – 17.00
Public lecture sponsored by EDAB (In Polish)
Tomasz Gabryelewicz (Mossakowski Medical Research Centre PAS, Warszawa, Poland)
Demencja i depresja – czy starość musi tak wyglądać?
(Dementia and Depression – whether the senescence has to look like that?)
Introduced by: Teresa Zalewska
Septemeber 1st-12th
EXHIBITION “The History of Polish Neuroscience”
Location: Museum of the University of Warsaw, Pałac Kazimierzowski, Krakowskie Przedmiescie
26/28
during the Congress the posters on display will be presented by the authors at a POSTER
SESSION III on Thursday and Friday, from 15.30 to 16.30.
Plenary Lectures 273
PLENARY LECTURES
L1
TARGETING OF mRNA TO POSTSYNAPTIC SITES
ON NEURONAL DENDRITES
Steward O.
Reeve-Irvine Research Center, University of California at Irvine,
Irvine, CA, USA
A fundamental aspect of gene expression in neurons involves delivery of certain mRNAs to synaptic sites on dendrites, where they
are locally translated at synapses. Local translation of mRNA plays
a critical role in synaptic consolidation, the process through which
early transient changes in synaptic efficacy become more enduring.
Mechanisms of transport and localization have been revealed through
studies of a unique immediate early gene (IEG) called Arc (activity-regulated cytoskeleton-associated protein), AKA Arg 3.1. Arc is
strongly induced by intense neuronal activity, but is unique amongst
known IEGs because its mRNA is rapidly delivered into dendrites. A
remarkable feature of Arc is that newly-synthesized Arc mRNA localizes selectively at active synapses. Live cell imaging studies using
a GFP-based labeling system reveal that exogenously expressed Arc
constructs assemble into particles that are transported at rates up to
65 μm/minute, which would allow the delivery of an mRNA from the
nucleus to synapses on distal dendrites within minutes. Transcripts
that contain the 3’UTR of Arc localize in a highly selective manner at
the base of dendritic spines, indicating that the signals mediating both
dendritic transport and synaptic targeting are present in the 3’UTR of
Arc mRNA. Docking of Arc mRNA at active synapses depends on
NMDA receptor activation, a rapid polymerization of actin in spines,
and local activation of MAP kinase. Continued strong activation of
synapses after Arc mRNA is docked at synapses triggers Arc mRNA
degradation; the mechanisms underlying this activity-dependent
mRNA degradation remain to be identified. I will discuss how this
mechanism might play a role in stabilizing changes in synapses induced by activity.
L2
MAKING MAP OF THE MIND: MOLECULAR
MECHANISMS OF NEURONAL MIGRATION
Rakic P.
Department of Neurobiology & Kavli Institute for Neuroscience,
Yale University School of Medicine, New Heaven, CT, USA
The identity, synaptic relationship and, ultimately, function of neurons
is defined by their position. It is particularly evident in the cerebral
cortex where neurons acquire their position by active migration before birth from multiple sites of origin and involve complex molecular
events and cell–cell interactions. We have used a variety of in vitro
and in vivo assays show how specific genes, encoding signaling and
morphoregulatory molecules and their receptors cooperate in orchestrating various components of migration such as the mode of neuronal
proliferation, phenotype determination, establishment of polarity, detachment from the local substrate and rate of migration to the proper
position in the cortex. Interestingly, some of these genes and molecules
are also implicated in the pathogenesis of cognitive disorders such as
schizophrenia, mild mental retardation, childhood epilepsy and autism.
In addition, we found that the rate of neuronal migration and precise
inside-to-outside sequence of their deployment can be disrupted by exposure of embryo to various physical, chemical, and biological agents.
Disruption or even slowing down of neuronal migration by either genetic or environmental factors results in gross heterotopias or in subtle
abnormalities of neuronal positions that eventually affect the pattern of
synaptic circuits and ultimately may cause variety of idiopathic disorders of highest cognitive function.
L3
ALTERNATIVE METHODS OF NEUROPROTECTION
IN BRAIN ISCHEMIA
Lazarewicz J.
Department of Neurochemistry, Mossakowski Medical Research
Centre PAS, Warszawa, Poland
Learning of cellular and molecular mechanisms of the ischemic neuronal damage led to development of pharmacological methods of
neuroprotection targeted at these mechanisms. They were effective in
animal stroke models, but none of them passed clinical trials. Thus the
alternative experimental neuroprotective strategies emerge. Preclinical
studies demonstrated that mild hypothermia significantly improves the
outcome in the animal models of stroke and brain injury. This method
of still unclear mechanism has been successfully used in specific clinical conditions and has been tested in several trials. It is hoped that moderate hypothermia soon may be introduced as an alternative method
of stroke treatment. Ischemic preconditioning is a way of inducing
tolerance to brain ischemia by preceding the injurious ischemia with
the sub-lethal stressors like short ischemia, mild hypoxia, heating or
pharmacological treatment. The exact mechanisms of activation and
induction of brain tolerance to ischemia by preconditioning are not
clear. Recently post-conditioning, i.e. neuroprotective effect of the
post-treatment with sub-lethal stressors after injurious ischemia has
been demonstrated, pointing to a therapeutic potential of such a treatment, still being tested at the preclinical level.
L4
GLIA AND GLUTAMATE: NEW KEY PLAYERS IN THE
PATHOLOGY OF MAJOR DEPRESSION
Rajkowska G.
Department of Psychiatry and Human Behavior, University of
Mississippi Medical Center, Jackson, USA
Postmortem studies in depression reveal age-dependent cell pathology
in prefrontal cortex. Prominent reductions in glia and specifically, in
astrocytes, are observed in younger depressed, whereas neuronal pathology is found in elderly with depression. As astrocytes regulate extracellular concentrations of glutamate (via glial glutamate transporters), an early deficit in astrocytes could lead to increases in extracellular
glutamate and toxic damage to neurons as depression progresses. This
is supported by postmortem studies of reduced expression of mRNA
and protein for the glial glutamate transporter in younger depressed and
by reductions in glutamatergic neurons in elderly depressed. Moreover,
alterations in glutamate metabolism are reported in neuroimaging studies of depressed patients. Interestingly, agents increasing expression of
glial glutamate transporters and/or altering glutamate neurotransmission show antidepressant activity. Our hypothesis that glial pathology
is an initial stage of cellular and neurochemical changes in depression
was confirmed by observations of glial/glutamate deficits in chronically stressed rodents. Pharmacologically-induced loss of astroglia,
but not neurons in the rat prefrontal cortex will induce depressive-like
behaviors. Moreover, treatment with riluzole (a modulator of glutamate
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release) reverses stress-induced depressive-like behaviors and blocks
glia impairments providing a link between dysfunction of glia and glutamate in depression.
L5
ALCOHOLISM: FROM NEUROBIOLOGY TO NEW
TREATMENT APPROACHES
Samochowiec J.
Department of Psychiatry, Pomeranian Medical University,
Szczecin, Poland
Great strides have been made in the last decade regarding the
importance of both genetic and environmental contributors
to the risk for heavy drinking, alcohol problems, and alcohol
use disorders. This lecture reviews recent research fi ndings regarding important endophenotypes contributing to these risks,
(e.g. low response to alcohol, personality traits, sweet liking).
Genetic fi ndings will highlight several genes that appear to
contribute to the level of response to alcohol, sweet liking and
some personality traits predisposing to alcohol seeking behavior. This presentation will review ongoing steps to identify additional genes in several large population studies. The lecture
will also briefly describe neurobiological background of drugs:
(1) registered for alcohol treatment – disulfi ram, naltrexon,
acamprosate, (2) off label drugs used in alcohol dependence:
topiramat and ondasetron and (3) new medication: nelmelfene.
Also, the approach of using the existing alcohol typologies
(proposed by Cloninger and Lesch) in treatment proposal will
be presented using our own data. Supported by MNiSW grant
nr N N402 189335
L6
REGULATION OF SYNAPTIC FUNCTION BY
MODIFICATION OF POSTSYNAPTIC DENSITY
PROTEINS
Sheng M.
Department of Brain and Cognitive Sciences, Massachusetts Institute
of Technology, Cambridge, MA, USA; Department of Neuroscience,
Genentech Inc, South San Francisco, CA, USA
Phosphorylation regulation of postsynaptic density proteins is
likely to be a major means of regulating synaptic function. The
PSD scaffold PSD-95, a powerful determinant of synaptic
strength, is a case in point. Its precise role during synaptic
plasticity (LTP versus LTD) has not been easy to interpret
from overexpression, RNAi or knockout mice experiments.
We found that the PSD scaffold PSD-95 is phosphorylated on
multiple sites in cultured neurons and in vivo. Ser-295 phosphorylation, mediated by a Rac1-JNK1 MAP kinase pathway
and countered by phosphatases PP1 or PP2A, promotes PSD-95
accumulation in synapses and is associated with LTP-inducing
stimuli. More strikingly, LTD-inducing stimuli causes dephosphorylation of ser-295 rapidly and profoundly, correlating with
activation of PP1. In addition, LTD was associated with phosphorylation of an N-terminal residue of PSD-95 by the protein
kinase GSK3b. This site is also bidirectionally modulated by
activity. A phospho-mimicking mutant of PSD-95 (S295DPSD-95; which cannot be “dephosphorylated”) impaired the
internalization of AMPA receptors in cultured neurons and
blocked the induction of LTD in cultured hippocampal slices.
Our data indicate that dephosphorylation of PSD-95 on ser295, and phosphorylation of the N-terminus of PSD-95, is required for mobilization of PSD-95 from the PSD, de-anchoring of AMPA receptors from the PSD for internalization, and
hence induction of LTD.
L7
CYTOKINE-MEDIATED CROSS-TALK BETWEEN
GLIA AND NEURONS: NEUROPATHOLOGICAL
IMPLICATIONS FOR EPILEPSY
Vezzani A., Balosso S., Maroso M., Rizzi M., Noè F., Zardoni D.,
Frasca A., Ravizza T.
Department of Neuroscience, Mario Negri Institute for
Pharmacological Research, Milan, Italy
Recent findings in experimental models and in the clinical setting
highlight the possibility that inflammatory processes in the brain
contribute to the etiopathogenesis of seizures and to the establishment of a chronic epileptic focus. Prototypical inflammatory cytokines such as IL-1β and TNF-α are overexpressed in epileptogenic
brain areas, prominently by glia and to a lesser extent by neurons
and endothelial cells of the blood brain barrier. Cytokine receptors
are also upregulated, and the related intracellular signalling is activated, highlighting both autocrine and paracrine actions of cytokines in diseased brain. Cytokines can profoundly affect neuronal
network excitability, and the recent demonstration of molecular and
functional interactions between cytokines and classical neurotransmitters, such as glutamate and GABA, provides one mechanism by
which cytokines affect neuronal activity. These interactions may
result in increased tissue excitability leading to seizures and cell
loss. These findings describe novel communications between glia
and neurons which may contribute to pathological conditions (e.g.
seizures, neurodegeneration) characterized by the activation of inflammatory processes, thus highlighting potential new targets for
therapeutic intervention.
L8
EXPLORING AND MANIPULATING FUNCTION
OF MICROGLIA IN BRAIN PATHOLOGIES
Kaminska B.
Department of Cell Biology, Nencki Institute of Experimental
Biology PAS, Warszawa, Poland
Microglia are multifunctional immune cells of the brain executing various functions and rapidly responding to pathological
insults. Brain injury, hypoxia, infection or aberrant protein accumulation may lead to chronic inflammation with a progressive
shift in microglia function towards inflammatory phenotype and
accumulation of immune cells. Under pathological conditions,
the interplay of extrinsic signals directs microglia towards neuroprotective or detrimental phenotype. Molecular mechanisms
of initiation, progression and termination of microglia-initiated
inflammatory responses in the brain, in particular gene networks
and signaling pathways are poorly understood. Characterization
of the global transcriptome of microglia exposed to inflammatory or cytoprotective signals and analysis of signalling pathways
revealed differences in expression of genes encoding cytokines/
chemokines and transcription regulators. Identification of signal-
Plenary Lectures 275
ling pathways contributing to discrete microglia phenotypes and
discovery of transcription regulators which may serve as “master
switches” for induction of an inflammatory phenotype, will allow
to target specific functions of microglia. Therapeutic approaches
targeting signal transduction in microglia will be discussed. A
greater understanding of microglia functions coupled with advances in pharmacology and gene therapy will support development of functionally “engineered” microglia able to convey neuroprotection.
L9
GROWTH FACTOR GENE DELIVERY FOR
ALZHEIMER’S DISEASE: FROM ANIMAL MODELS
TO CLINICAL TRIALS
Tuszynski M.1, Nagahara A.1, Schroeder B.1, Coppola G.2,
Wang L.1, Blesch A.1, Rockenstein E.1, Geschwind D.2, Masliah E.1,
Koo E.1, Chiba A.1
1
Department of Neuroscience, University of California, San Diego,
CA, USA; 2 Department of Neurology, University of California
Los Angeles, CA, USA
Nervous system growth factors have extensive effects on neuronal
function and survival. Nerve growth factor (NGF) prevents the
death and stimulates the function of basal forebrain cholinergic
neurons in correlational models of Alzheimer’s disease (AD), leading to its translation to Phase 1 and 2 human clinical trials. Separately, Brain-Derived Neurotrophic Factor (BDNF) influences the
survival and function of entorhinal cortical and hippocampal neurons in several animal models of AD, including transgenic mutant
APP-expressing mice; aged rats and lesioned rats; and aged and
lesioned primates. These beneficial effects appear to occur independent of alteration in beta amyloid load. We are currently examining the extended safety and tolerability of BDNF gene delivery
to the entorhinal cortex in additional animal studies, potentially
leading to specific targeting of short term memory loss in future
human AD trials. This talk will review the history and current
status of growth factor gene delivery in AD and other neurodegenerative disorders.
EJN-lecture
THETA BURST STIMULATION OF DORSOLATERAL
PREFRONTAL CORTEX REVEALS HEMISPHERIC
ASYMMETRY IN STRIATAL DOPAMINE RELEASE
DURING SET-SHIFTING TASK IN HUMAN
Ko J.1, Monchi O.2, Ptito A.1, Bloomfield P.3, Houle S.3,
Strafella A.4
1
Montreal Neurological Institute, McGill University, Montreal,
Canada; 2 Functional Neuroimaging Unit, University of Montréal,
Montreal, Canada; 3 PET Imaging Centre, Centre for Addiction and
Mental Health, Toronto, Canada; 4 Toronto Western Research Institute
and Hospital, University of Toronto, Toronto, Canada
Previous neuroimaging studies have shown that executive processes requiring planning and set-shifting [e.g. Montreal card
sorting task (MCST)] may engage the dorsolateral prefrontal
cortex (DLPFC) while inducing dopamine (DA) release in the
striatum. However, functional imaging studies can only provide neuronal correlates of cognitive performance and cannot
establish a causal relation between observed brain activity and
task performance. In order to investigate the contribution of the
DLPFC during set-shifting and its effect on the striatal DAergic
system, we applied continuous theta burst stimulation (cTBS)
to left and right DLPFC. Our aim was to transiently disrupt its
function and to measure MCST performance and striatal DA
release during [11C]raclopride PET. cTBS of the left DLPFC impaired MCST performance and DA release in the ipsilateral caudate–anterior putamen and contralateral caudate, as compared
to cTBS of the vertex (control). These effects were limited only
to left DLPFC stimulation but not right DLPFC. This is the fi rst
study showing that cTBS, by disrupting left DLPFC function,
may indirectly affect striatal DA release during performance of
executive tasks. This cTBS-induced regional prefrontal effect
and modulation of the frontostriatal network may be important
for understanding the contribution of hemisphere laterality and
its neural bases with regard to executive functions, as well as
for revealing the neurochemical substrate underlying cognitive
deficits.
EDAB lecture (in Polish)
DEMENTIA AND DEPRESSION – WHETHER THE
SENESCENCE HAS TO LOOK LIKE THAT?
Gabryelewicz T.
Department of Neurodegenerative Disorders, Mossakowski Medical
Research Centre PAS, Warszawa, Poland
Dementia is a neurological condition manifested by reduced
cognitive and psychological functioning, altered behaviour and
decreased autonomy for social and professional performance in
activities of daily living. Dementia is one of the most important
causes of disability in the elderly. The number of dementia patients is increasing, as the population is growing older. Recent
epidemiological evidence suggested a 2001 prevalence of 24.3
million cases of dementia worldwide. Alzheimer’s disease (AD)
remains the most common cause of dementia, responsible for
60–70% of cases in Europe. There are 4.6 million new cases of
dementia reported every year – that’s one new case every 7 seconds. Patients with AD gradually lose the ability to function independently and interest in life and many experience changes in
personality, social withdrawal, uninhibited behaviour, and psychotic symptoms such as delusions, hallucinations and aggressive behaviour. The associated likely impact on the health and
quality of life of persons with AD, their families, and societal
healthcare and welfare resources, have led many authors to describe the condition as a “ticking bomb”. The depression prevalence in later life is 8 to 15%. Suicide in the elderly represents
an immensely important and often overlooked medical problem.
Based on its many distinct features, age-specific risk factors
could be identified. Proper diagnosis and treatment of affective
disorders – contributing greatly to the pathogenesis of suicide in
the elderly – could lead to a major decline in the suicide rate in
this population.
276
SYMPOSIUM I
Learning-Dependent Brain Plasticity:
Systems View
SI.1
SYNAPTIC MECHANISMS OF SENSORY PERCEPTION
Petersen C.
Laboratory of Sensory Processing, Brain Mind Institute, Faculty of
Life Science, Ecole Polytechnique Federale de Lausanne (EPFL),
Lausanne, Switzerland
A key goal of modern neuroscience is to understand the neural circuits
and the synaptic mechanisms underlying simple forms of sensory perception and associative learning. Here, I will discuss our efforts to
characterise sensory processing in the mouse barrel cortex, a brain
region known to process tactile information relating to the whiskers
on the snout. Each whisker is individually represented in the primary
somatosensory neocortex by an anatomical unit termed a “barrel”.
The barrels are arranged in a stereotypical map, which allows recordings and manipulations to be targeted with remarkable precision. In
this cortical region it may therefore be feasible to gain a quantitative
understanding of neocortical function. As a mouse explores its environment, the whiskers are actively moved backwards and forwards as
if searching the space for tactile input. Indeed sensory information
in this pathway is usually actively acquired. It is therefore crucial to
measure and manipulate cortical function in awake behaving mice.
We have begun this process using whole-cell recordings, voltage-sensitive dye imaging, viral manipulations and two-photon microscopy.
Through combining these techniques with behavioural training, our
experiments provide new insight into sensory perception at the level
of individual neurons and their synaptic connections.
SI.2
REORGANIZATION OF THE VISUAL CORTEX
FOLLOWING MONOCULAR CIRCUMSCRIBED
RETINAL LESIONS: UPDATE ON HEBBIAN LEARNING
RULES
Waleszczyk W.1, Wang C.2, Bardy C.2, Calford M.3, Dreher B.4
1
Department of Neurophysiology, Nencki Institute of Experimental
Biology PAS, Warszawa, Poland; 2 School of Medical Sciences,
The University of Sydney, NSW, Australia; 3 Faculty of Health,
The University of Newcastle, NSW, Australia; 4 School of Medical
Sciences, ARC Centre of Excellence in Vision Science,
The University of Sydney, NSW, Australia
Following circumscribed monocular retinal lesions most cells located
in the lesion projection zone (LPZ) in the cat’s primary visual cortices
remain binocular but their receptive fields (RFs) revealed by stimulation via the lesioned eye, are displaced into proximity of the lesion.
The process of topographic reorganization (adaptive learning) of the
visual cortex, is based on spike timing-dependent plasticity rather
than the classical Hebbian rules [Young et al. (2007) Nat Neurosci
10: 887–895]. The contrast sensitivities of the classical RFs of LPZ
neurons revealed by stimulation via the lesioned eye are significantly
lower, while the suppresive influences of their “silent”, extra-classical
RFs are weaker than those of their counterparts revealed by stimulation via non-lesioned eye. Other properties (e.g. orientation, spatial
and temporal frequency preferences) however match well the proper-
ties of RFs revealed by stimulation via non-lesioned eye. These similarities are presumably visual experience-dependent, that is, they are
related to the fact that in the post-lesion period (from adolescence to
maturity), the neuronal activities originating from each retina are correlated by effectively synchronized exposure to the same visual stimuli. These results are consistent with the hypothesis that the ectopic
RFs are based on enhancement of the synaptic efficacy of long-range,
excitatory intracortical connections. Financial support: Nencki Institute, Poland; NHMRC and ARC grants, Australia.
SI.3
WHAT IS STORED IN THE HIPPOCAMPUS DURING
A TACTILE CATEGORIZATION TASK?
Diamond M., Itskov P., Vinnik E.
Department of Cognitive Neuroscience, International School for
Advanced Studies, Trieste, Italy
To understand how sensory experiences are stored in the brain, we
examined neuronal firing in hippocampus during tactile behavior.
Rats learned to associate stimulus texture with reward location;
multiple textures were associated with the same reward location
and thus formed a behavioral category. Rapid firing rate modulation carried texture identity information (10% of neurons), free
from spatial and behavioral confounds; slow firing rate modulation carried behavioral category information (63% of neurons).
Category information appeared during texture contact, simultaneous with an increase in theta power in the local field potential; it
persisted or recurred during reward collection, when theta power
was suppressed and “reward neurons” (8%) fired. Reward-triggered recurrence of category information could be a mechanism
to link stimulus, action, and outcome when separated in time.
SI.4
THE LEARNING BRAIN – ACTIVATION DURING
CLASSICAL CONDITIONING
Kossut M., Cybulska A.
Department of Molecular and Cellular Neurobiology, Nencki Institute
of Experimental Biology PAS, Warszawa, Poland
The neural bases of appetitive and aversive conditioning are different,
and at various stages of learning may engage distinct cortical and subcortical networks. Using [14C]2-deoxyglucose (2-DG) autoradiography we examined brain activation in mice during classical conditioning involving stimulation of whiskers on one side of the muzzle paired
with an aversive or appetitive UCS. Both variants result in modification of cortical representations of vibrissae activated during the conditioning. Analysis of autoradiograms revealed that the nucleus basalis of Meynert (NBM) and ventral pallidum showed stronger labeling
during appetitive training while the lateral hypothalamus (LH) and
basolateral amygdala (BLA) were activated only by aversive learning. Apart from that, classicalconditioning with appetitive or aversive UCS increased 2-DG uptake in a similar set of brain structures
– the posterior parietal cortex (PPC), cingulate (CG) and retrosplenial
gyrus (RET), caudate nucleus (CPU) and nucleus accumbens (NA).
Formation of sensory association, compared to pseudoconditioning,
induces more activity in the subcortical sensory processing pathway
(ventral postero-medial and posterior nuclei of the thalamus) but not
in the barrel cortex. Also, conditioning contrasted with pseudoconditioning increases activity in structures important for cognitive and attentional functions (PPC, NA, CG, RET, CPU), which might provide
the enhancing input necessary for memory trace formation.
Symposia 277
SYMPOSIUM II
Using Neuroimaging to Understand
Human Brain Function
SII.1
INSIDE BOLD – WHY NEURONAL ACTIVITY
MODULATES MR SIGNAL?
Bogorodzki P.
Institute of Radioelectronics, Warsaw University of Technology,
Warszawa, Poland
Although fMRI methods are well recognized as a powerful tool
in neuroimaging, they still suffer from lack of repeatable quantitative measures of effect studied, especially in disease group studies,
where pathologically changed brain morphologies or distortions
caused by head movement arise. In such cases where typical statistical parametric mapping (SPM) approach based on general linear
model (GLM) modeling fails, additional methods, giving complementary measures to standard statistical maps SPM may comprise
an alternative approach. This subject covers two basic methodologies, which will be presented in this presentation: (1) A novel
technique called Mean Regional Response (MRR) uses features
derived from time-intensity curves in anatomically or functionally
defined regions of interest (ROIs). Resulting features can be used
for higher level analysis, like ANOVA or highlight differences between groups of subjects allowing inference about group separation; (2) Structural Equation Modeling (SEM) technique is focused
on the extraction of connections between the active regions. Although SEM still do not allow arbitrary connection patterns, its
use allows determination of time varying and bi-directional, asymmetric connections for groups of interest (patient vs. controls etc.).
A methodological basics of BOLD modeling, which can be considered as a basis for most detection and classification methods, will
be presented and illustrated with author’s fMRI studies.
SII.2
NEURAL BASES OF PRAXIS AND TARGET-DIRECTED
ACTIONS
Kroliczak G.
Department of Psychology, University of Oregon, Eugene, OR,
USA
Neuroimaging evidence (Kroliczak and Frey 2009) indicates that,
in right-handers, the left-hemispheric praxis representation network
(PRN) constitutes a general domain neural substrate for the planning
of meaningful actions, whether or not they involve objects. Less is
known about the relationship between PRN and motor dominance
in consistent left-handers. Nevertheless, more recent neuroimaging
in healthy left-handed adults also shows that planning either transitive/tool use pantomimes or intransitive gestures is associated with
increases of activation in the same cortical regions. In addition to
the left parietal cortex, however, these increases tend to be more
bilateral and involve the dorsal premotor and pre-supplementary
motor cortices, as well as the rostral middle frontal gyri. These data
suggest that despite reversed motor dominance and more bilateral
PRN, planning any meaningful actions might be represented in a
common network. The results will be discussed in the context of
cortical areas mediating real, target-directed actions.
SII.3
THE NEURAL CORRELATES OF LANGUAGE
PROCESSING AND ITS DISORDERS OVER THE LIFE
SPAN
Heim S.
Department of Psychiatry and Psychotherapy, RWTH Aachen
University, Aachen, Germany
I fi rst present the results of a number of fMRI studies investigating the left-lateralised language network in the human brain.
The focus will be here on phonological and lexical processing,
which both recruit Broca’s region. These data are summarised
in a model which serves as framework for the subsequently
presented data. Next, I explore commonalities and differences
between primary school children and adults with respect to
lexical and phonological processing. In the children, the same
networks are involved, but are less specialised as compared
to adults. Then, neurocognitive differences between dyslexic
and normally reading children for lexical and phonological
processing as well as for other cognitive functions relevant for
reading are discussed. These functions include auditory and
visuo-magnocellular processing, attention, and automatisation. Multivariate testing of these functions reveals different
cognitive subtypes of dyslexia. Consequently, a new training
approach is outlined. With respect to cognitive functions relevant for reading, fMRI reveals consistent right-hemispheric
involvement for dyslexic children, which is regarded to reflect
compensatory processes. Finally, a transfer is made to adult
dyslexic processing and to linguistic difficulties in vascular
and neurodegenerative aphasia in middle and older age. This
research is funded by BMBF 01GJ0613/01GJ0614/GJ01203907,
DFG HE 5204/3-1, and JARA-BRAIN Excellence Initiative.
SII.4
NEURAL CORRELATES OF FALSE RECOGNITION
Marchewka A.
The neuronal basis of false recognition is still a subject of extensive debate. We used voxel based morphometry (VBM) to examine structural brain correlates of false and correct recognition
processes. Since several studies indicate that emotional content
facilitates false recognition we employed emotional stimuli taken from International Affective Picture System (IAPS). Behavioral measures, i.e., true and false recognition rates were used as
covariants in VBM analyses. VBM results indicated that the true
recognition correlated positively with grey-matter (GM) density
in bilateral amygdala, anterior cingulate and middle temporal
gyrus, i.e., brain regions, involved in the memory of emotional
material, as revealed by fMRI results. False recognition correlated negatively with GM density in prefrontal areas (BA47 and
BA9), which were implicated in false recognition by our previous fMRI study. Taken together, these results support the role
of the prefrontal cortex in monitoring retrieval and limiting the
probability of false recognition. Our VBM fi ndings (1) point to
the brain structures critical for correct and false recognition of
emotional stimuli and (2) disclose structural differences between
these processes.
278
Workshop
Quantitative Analysis of Protein–Protein
Interactions
W1
NON-NEGATIVE MATRIX FACTORIZATION:
A NOVEL METHOD TO DETERMINE REFERENCE
SPECTRA OF FLUORESCENT DYES IN A CELLULAR
ENVIRONMENT
Neher E.
Department of Membrane Biophysics, Max-Plank-Institute for
Biophysical Chemistry, Munchen, Germany
All spectrally resolved fluorescence-based methods depend critically on the availability of accurate reference spectra. Very often
spectra in a cellular environment do not agree with those measured
in a cuvette. Also, for a given measurement instrument-specific
corrections have to be applied. We recently developed a method to
extract reference spetra from standard fluorescence image stacks,
such as those provided by a Zeiss Meta microscope. This method
also allows one to separate spectral components of several simultaneously present dyes on a pixel by pixel basis. It should be ideal
to complement lux-FRET measurements, providing reference
spectra in the presence of autofluorescence.
W2
QUANTITATIVE MEASUREMENTS OF cAMP
CONCENTRATION WITH A NEW EPAC BASED
FRET-SENSOR
Richter D.
Department of Neuro and Sensory Physiology, University of
Göttingen, Göttingen, Germany
FRET-based biosensors can be used for the analysis of intracellular
signalling, including sensors for monitoring cyclic AMP. The concept is that the donor/acceptor emission ratio excited at the donor
excitation wavelength can be used as a concentration dependent
measure in donor/acceptor 1:1 tandem biosensors. General problem,
however, is that this ratio varies not only with the changes in cAMP
concentration, but also with the changes of the ionic environment
or other factors affecting the folding probability. We are developing a novel Epac Based FRET-Sensor that is much less sensitive to
changes in ion concentrations to obtain a reliable measure of the
absolute cAMP concentrations under patho-/physiological conditions with high temporal and spatial resolution. Such data will be
correlated with electrophysiological measurements obtained under
pathological as well as physiological conditions.
W3
STIMULATION-DEPENDENT CHANGES IN
OLIGOMERIC CONFORMATION OF SEROTONIN
5-HT1A RECEPTORS
Ponimaskin E.
Department of Cellular Neurophysiology, Hannover Medical School,
Hannover, Germany
An increasing number of G protein-coupled receptors (GPCRs)
have been shown to form oligomeric units in the plasma membrane. In many cases there is evidence that this oligomerization
can result in altered receptor pharmacology, desensitization and
trafficking. These alterations can consequently result in different
effects in intracellular signalling, thus diversifying the functional role of a GPCR. The serotonin (5-HT)1A receptor is a GPCR
involved in multiple processes, among which, the regulation of
neurogenesis, respiratory control, as well as depression and anxiety states have found the most interest. Biochemical experiments
performed in N1E-115 neuroblastoma cells have suggested that
5-HT1A receptors form homooligomers. Acceptor-photobleaching
allows for the direct measurement of apparent FRET between the
fluorophores of CFP- and YFP-tagged proteins, further suggesting some specific interaction between the receptors. Furthermore,
with the use a novel FRET quantification method, we have also
been able to discriminate between specific and random proteinprotein interaction and to verify dynamics of homo- and heterooligomerization between 5-HT1A and 5-HT7 receptors.
W4
LUX-FRET METHOD FOR QUANTITIVE ANALYSIS
OF FRET SIGNAL
Wlodarczyk J.
A novel method for spectral analysis of FRET-signals in living
cells will be presented. The method allows for the determination
of exact stoichiometry of interacting molecules as well as apparent FRET efficiency from two-wavelength measurement. Further
considerations allow us to predict the influence of incomplete labeling of interacting partners. The method is based on information
obtained from the full fluorescence emission spectra. In addition
our analysis explicitly takes into account the contributions from
simultaneously present free donors, acceptors and FRET-pairs, as
well as the effects of non-functional fluorophores. The determination of apparent FRET efficiency in our method does not require,
as in some other methods, acceptor photobleaching experiments,
fluorescence lifetime measurements nor cell fixation. The method
presented here can be directly applied to individual pixels of a
fluorescence image in fluorescence microscopy image analysis.
W5
HIGH-RESOLUTION LIGHT-MICROSCOPIC STUDIES
OF SYNAPTIC EXTRACELLULAR PROTEOLYSIS
Wilczynski G.
Proteolytic remodeling of perisynaptic environment, including
extracellular matrix and adhesion molecules, is a novel important
mechanism of synaptic plasticity. Among several enzymes participating in the phenomenon, the major role is played by matrix metalloproteinase 9 (MMP-9), a principal neuronal MMP. Recent evidence indicates MMP-9 being involved in long-term potentiation,
learning and memory formation, as well as in pathological plasticity underlying epileptogenesis. Here, I present the results of the
light-microscopic studies on MMP-9 localization at the synapses in
situ, in both central and peripheral nervous systems. The data have
been obtained using fluorescent cytochemical techniques, including immunoflorescence, in situ hybridization and in situ zymography, with the aid of deconvolution-enhanced confocal microscopy.
The results underscore the potential of light-microscope-based molecular morphology for studying synaptic function in situ.
Symposia 279
SYMPOSIUM III
Learning-Dependent Brain Plasticity:
Synaptic View
SIII.1
SUBREGIONAL SPECIFIC ROLE OF HIPPOCAMPAL
SYNAPTIC PLASTICITY IN SPATIAL MEMORY
PROCESSING: SIGNIFICANCE OF β-ADRENERGIC
RECEPTORS
Manahan-Vaughan D.
Department of Experimental Neurophysiology, Medical Faculty, Ruhr
University, Bochum, Germany
Neural mechanisms for spatial memory formation are believed
to comprise an integration of processes mediated by hippocampal synaptic plasticity in the form of long-term potentiation
(LTP) and long-term depression (LTD). Novel space consists
of several types of information, but that may evoke differential responses in individual hippocampal subregions. In CA1,
CA3 and dentate gyrus (DG) subregions and at multiple types
of hippocampal synapses (perforant path-DG, mossy fiber-CA3,
Schaffer collateral-CA1) exploration of a novel empty environment facilitates the expression of robust LTP (>24 h) in freely
behaving animals. In contrast, LTD facilitation is subregionand synapse- specific and dependent on the nature of the cues.
In the CA1 region, partially concealed, small, contextual cues
facilitate LTD. LTD in DG is facilitated by large directional
cues. Arousal, mediated by activation of the noradrenergic
(NA) system is a critical factor in information acquisition and
may enhance the encoding of novel spatial information. We observed that learning-facilitated induction of LTD is inhibited
by β-adrenoreceptor antagonist, and facilitation of LTD and the
encoding of spatial memory is elicited when the locus coeruleus
is activated. These results suggest that β-adrenoreceptors may
facilitate encoding of spatial information through synaptic plasticity in the hippocampus and that NA released by the locus
coeruleus during novel exploration, is a key factor in effective
information acquisition.
SIII.2
SENSORY LEARNING ENHANCES GABAERGIC
SYNAPTIC TRANSMISSION IN THE BARREL CORTEX
OF THE MOUSE
Tokarski K.1, Urban-Ciecko J.2, Kossut M.2, Hess G.1
1
Institute of Pharmacology PAS, Kraków, Poland; 2 Department
of Molecular and Cellular Neurobiology, Nencki Institute of
Experimental Biology PAS, Warszawa, Poland
The barrel cortex is part of rodent primary somatosensory cortex, engaged in processing tactile information from the vibrissae. This part of the cortex contains layer IV cytoarchitectonic
units – barrels, each corresponds to one of the contralateral
vibrissae. Short-term, classical conditioning paradigm, consisting of three daily sessions of tactile stimulation of a row
of vibrissae paired with electrical shock to the tail, induces
expansion of the representation of stimulated vibrissae, pronounced in layer IV of adult mice barrel cortex. This effect
has been accompanied by elevation in mRNA and protein level
of GAD67, selective up-regulation of GABA and GAD67 in
neurons within centres of the barrels receiving inputs from
stimulated vibrissae, and an increase in the number of GADimmunoreactive puncta. To investigate whether classical conditioning affects synaptic transmission in layer IV of the barrel
cortex we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) using ex vivo brain slices prepared from adult
mice previously subjected to conditioning. We show that this
associative learning paradigm results in a selective increase in
the frequency of sIPSCs in layer IV excitatory neurons located
within the barrel representing stimulated vibrissae, evident 24
h after the end of training. These data indicate that aversive
training results in a selective and long-lasting enhancement of
GABAergic transmission within the cortical representation of
stimulated vibrissae.
SIII.3
IMPACT OF SENSORY LEARNING ON GABA-ERGIC
TONIC INHIBITION IN MOUSE BARREL CORTEX
Mozrzymas J., Urban-Ciecko J.
Laboratory of Neuroscience, Department of Biophysics, Wroclaw
Medical University, Wrocław, Poland
Sensory learning is known to affect neuronal function, morphology and synaptic connectivity. It has been shown that various forms of classical conditioning involving whisker stimulation induce morphological changes in respective barrels
(altered expression of GABAARs, NMDARs and GAD67).
More recent studies have shown that sensory learning enhanced the inhibitory synaptic transmission (Tokarski et al.
2007). Our aim was to follow up the impact of sensory learning on GABAergic currents in the barrel cortex by addressing
the issue of tonic GABAergic inhibition. We have characterized the tonic currents in naive mice for different cell types
(Regular Spiking, Regular Spiking Non-Pyramidal and Fast
Spiking) and found that the values of currents are correlated
with the neuronal cell type. Interestingly, classical conditioning (tactile whisker stimulation associated with electric foot
shock) affected the value of tonic current in the cell specific
manner. To further explore the underlying mechanisms, we
have used a specific “superagonist” (THIP) for δ-subunit containing GABAA receptors. Our data indicate that observed
here impact of sensory learning on tonic currents results from
altered responsiveness of δ-subunit containing receptors. In
conclusion, we provide evidence that behavioral training does
affect the tonic inhibition in the barrel cortex. Supported by
the Ministry of Science and Higher Education grants PBZ/
MNiSW/07/2006/02 and N401 028 32/0664.
SIII.4
A TRANSIENT SWITCH IN THE SIGN OF NMDARDEPENDENT SYNAPTIC CHANGE DURING
EXPERIENCE-DEPENDENT PLASTICITY IN VIVO
Barth A., Weng J.
Department of Biological Sciences and Center for the Neural Basis
of Cognition, Carnegie Mellon University, Pittsburgh, PA, USA
Sensory experience can augment excitatory synaptic strength.
Removal of all but one whisker from both sides of the mouse
280
face (single-whisker experience; SWE) leads to an increase in
synaptic strength at layer 4-2/3 synapses as well as elevated
whisker-evoked fi ring in vivo. Although this potentiation requires NMDAR-activation in its early stages, NMDAR-activation subsequently shift the sign of synaptic change to favor
synaptic depression. Thus, timed administration of NMDARantagonists can facilitate experience-dependent increases in
excitatory synaptic strength. Here we show that the transition
to NMDAR-dependent LTD is transient during ongoing experience-dependent synaptic plasticity in the barrel cortex. Systemic injection of NMDAR-antagonists within the fi rst 12 h of
SWE yields a robust enhancement of synaptic strength at layer
4-2/3 synapses. In contrast, later injection (after 36 h of SWE)
does not alter synaptic strength compared to vehicle-injected
control animals. Thus, as synaptic changes are consolidated by
longer periods of whisker plasticity, the window for NMDARdependent synaptic depression closes and strengthened synapses become resilient to biochemical cascades responsible for
weakening. Such a process may insure that spurious increases
in synaptic strength are not permanently incorporated into cortical networks.
SYMPOSIUM IV
Mechanisms, Functions and Modulation
of Central Nervous System Inflammation
SIV.1
MICROGLIAL SENESCENCE AND DEGENERATION
IN THE PATHOGENESIS OF NEURODEGENERATIVE
DISEASE
Streit W.
Department of Neuroscience, University of Florida, Gainesville, FL,
USA
The role of microglia in Alzheimer’s disease (AD) pathogenesis
remains unknown. Although many studies maintain that chronic
microglial activation in the AD brain is detrimental and contributes
to neurodegeneration, anti-inflammatory drugs show little promise for AD treatment or prevention. Studies provide support for the
microglial dysfunction hypothesis, which postulates that the neurofibrillary degeneration of AD is the result of declining microglial
neuroprotection resulting from aging-related microglial senescence
and degeneration. I will report histopathological findings from humans covering the spectrum from none to severe AD pathology,
including patients with Down syndrome, showing that degenerating neuronal structures positive for tau, invariably colocalized with
severely dystrophic rather than with activated microglial cells. Using Braak staging of AD neuropathology, I will demonstrate that
microglial dystrophy precedes the spread of tau pathology. Amyloid
deposits devoid of tau-positive structures are found to be colocalized with non-activated, ramified microglia, suggesting that amyloid does not trigger microglial activation. The findings indicate
that when microglial activation does occur in the absence of an
identifiable acute CNS insult, it is likely to be the result of systemic
infectious disease. These findings strongly argue against the belief
that neuroinflammatory changes contribute to AD dementia. They
may profoundly influence future treatment approaches.
SIV.2
MANIPULATING INFLAMMATION AND GLIOSIS
IN NEUROLOGICAL DISEASES – A CLINICAL
PERSPECTIVE
Czlonkowska A.1,2
1
2nd Department of Neurology, Institute of Psychiatry and Neurology,
2
Departament of Experimental and Clinical Pharmacology, Warsaw
Medical University, Warszawa, Poland
In many neurological diseases, an inflammatory reaction in the
brain is observed. In some of them, as in multiple sclerosis, the
brain inflammation is a result of a systemic autoimmune response.
In others, like the Parkinson’s disease, Alzheimer’s disease, brain
injury and stroke, an inflammation and glial proliferation in the
brain are a secondary phenomenon to neural degeneration or injury. The role of these reactions in the pathogenesis of neurological
diseases is unclear. From one side they can contribute to greater
neurological deficit. From the other side, inflammatory cells may
release trophic factors and contribute to neuroprotection and neuroregenaration. Experimental works provide some evidences that
pharmacological inhibition of brain inflammation and glial proliferation may be protective but also may be harmful. The influence
of anti-inflammatory therapies on the progression of neurological
diseases depends on a dose and time of the treatment after injury.
Therefore, a clinical use of drugs influencing inflammation and glial proliferation is very difficult. Despite many anti-inflammatory
therapies used in neurological diseases at present, there is no clinical evidence of their beneficial effects. Various immunomodulatory therapies have been tested in multiple sclerosis, but only some
modest modifications of clinical course are observed. Similarly, in
neurodegeneration like in the Parkinson’s disease, non-steroidal
anti-inflammatory drugs do not change disease progression.
SIV.3
GLIA AND INFLAMMATION IN THE PARKINSONIAN
SUBSTANTIA NIGRA
Graeber M.B1,2
1
King Fahad Medical City, Faculty of Medicine, Riyadh, Kingdom
of Saudi Arabia; 2Brain & Mind Research Institute, University of
Sydney, Sydney, Australia
Microglia are the resident sensors of pathology in the central
nervous system. Their main function is to control tissue damage
and to engage in tissue repair processes. The exact role of microglial cells in neurodegenerative disorders is unknown. Based
on their expression of some molecules found in established inflammatory disease states it has been suggested that microglia
exert tissue destructive functions in Parkinson’s disease (PD).
We have indeed observed a correlation between α-synuclein
(αSN) deposition and MHC class II-expressing microglia in the
substantia nigra in PD but it remains unclear whether there is a
pathogenetic link. In addition, in the absence of infiltrating Tcells which characterise chronic inflammatory or autoimmune
conditions, e.g. CNS infections and multiple sclerosis, classical
inflammation does not occur in PD. We therefore believe that
“(micro)glial inflammation” represents a distinct process. This
idea is supported by the up-regulation of CD163 by nigral microglia in PD suggesting a possible role of resident microglia
in the down-regulation of inflammatory activity. Astrocytes
in PD nigra strongly up-regulate their expression of the Hsp40
Symposia 281
chaperone, DnaJB6 and they also increase their production of
metallothioneins. They are thus in an activated state. The homocysteine-induced endoplasmic reticulum protein (Herp) may
serve as a molecular link between neurodegeneration and the
inflammation-like changes observed in PD.
SIV.4
MODULATION OF GLIA ACTIVATION IN
NEUROPATHIC PAIN
Przewlocka B.
Department of Pain Pharmacology, Institute of Pharmacology PAS,
Kraków, Poland
There is a growing body of evidence indicating that glial cells
have causal role in the pathogenesis of pain hypersensitivity
following nerve injury. We aimed to examine how chronic constriction injury (CCI) to the sciatic nerve influences glia activity
markers, cytokine levels and expression of mGlu receptors in the
spinal cord and dorsal root ganglia (DRG) as well as features
of the neuropathic pain, such as allodynia and hyperalgesia in
rats and mice. Our study showed that glial markers (C1q, GFAP),
interleukins (IL-1β, IL-6) and mGlu receptor (mGlu5, mGlu3)
mRNAs were strongly elevated ipsilaterally in the spinal cord
after CCI. Microglial cells were more activated in the spinal cord
in contrast to DRG where astrocytic activation prevailed. In the
ipsilateral DRG, IL-1α, IL-6, and IL-10 mRNA levels were increased. Western blot analysis showed the presence of IL-1α protein in the DRG and down-regulation of these proteins after CCI.
Minocycline and/or pentoxifylline administration reversed the
injury-induced changes in glia markers and mGlu receptor mRNAs and protein levels, and significantly attenuated CCI-induced
allodynia and hyperalgesia. The implication of glial activation
paralleled with upregulation of mGlu receptors in neurodegenerative processes suggests that pharmacological targeting of spinal
microglia in chronic pain may provide an effective therapy for
controlling clinical pain syndromes. Support: statutory funds of
the Institute of Pharmacology, PAS.
SYMPOSIUM V
Stress-Response Systems in the Pathogenesis
and Prognosis of Cancer, Inflammation
and Autoimmune Diseases
SV.1
STRESSOR EFFECTS ON IL-10 AND IFN-γ
PRODUCTION ARE MEDIATED BY β-ADRENERGIC
AND GLUCOCORTIOCID RECEPTORS AND
ATTENUATED BY CHLORDIAZEPOXIDE
Curtin N.1, Boyle N.1, Griffin E.1, Mills K.2, Connor T.1
1
Trinity College Institute of Neuroscience, 2 School of Biochemistry
& Immunology, Trinity College Dublin, Ireland
Research indicates that psychological stress can modulate immune responses. Here we demonstrate that restraint stress suppresses interferon (IFN)-γ production and signalling in mice
following in vivo lipopolysaccharide (LPS) treatment. Stress
also suppressed production of the IFN-γ inducing cytokine interleukin (IL)-12 and increased production of the anti-inflammatory cytokine IL-10, which can inhibit both IL-12 and IFN-γ
production. Studies with IL-10 knockout mice demonstrate that
IL-10 does not mediate the suppressive effect of stress on IFN-γ
production. Consistent with a role for glucocorticoids in the immunosuppressive actions of stress, the glucocorticoid receptor
antagonist mifepristone blocked the stress-related suppression
of IFN-γ. In contrast, the stress-induced increase in IL-10 was
blocked by the beta-adrenergic receptor antagonist nadolol. Addition of IL-12 to LPS-stimulated spleen cells reversed the suppressive effect of stress and corticosterone on IFN-γ production, suggesting that reduced IL-12 production is a key event
in stress-induced suppression of IFN-γ production. Finally, we
demonstrate that treatment with the anxiolytic drug chlordiazepoxide prevents the ability of stress to increase IL-10 and suppress IFN-γ production.
SV.2
STRESS, EMOTION, AND NATURAL KILLER CELL
ANTITUMOR ACTIVITY
Lewandowska D.
Department of Animal Physiology, University of Gdansk, Gdańsk,
Poland
Neoplasia is one of the paradigmatic psychoneuroimmunology
(PNI) disorders, and several studies showcased clinical associations between behavioral and emotional states and cancer
and between those states and the immune system. If clinically
relevant PNI effects on cancer were to be found, they would
likely depend on selection of appropriate parameters: tumors
susceptible to identified immunologic functions; immune
effects acting at appropriate points in the natural history of
neoplastic disease, and effective immunomodulatory intervention. Animal studies suggest that natural killer (NK) cells are
important for cancer control, especially with respect to dissemination and growth of metastasis. Surgical and psychological stress have been reported to suppress NK activity (NKCC)
in animals and humans and this suppression was shown to
compromise animal resistance to tumor progression. Studies
282
published by Ben-Eliyahu showed powerful model of stressrelated NK-dependent metastasis. Metastatic progression in
their MADB106 mammary adenocarcinoma model is related
to surgical stress, swim stress, hypothermia or social confrontation and stress hormone-induced suppression of NKCC that
is not dependent on physical dissemination of the primary tumor. Moreover, perioperative suppression of NKCC in the immediate postoperative period may contribute in part to the risk
of subsequent emergence of metastasis, and perioperative interventios that reduce this risk may improve patient outcome.
SV.3
NEURAL PATHWAYS SIGNALING CANCER AND
INFLAMMATION: INTERFACE WITH STRESSRESPONSIVE BRAIN REGIONS
Goehler L.
School of Nursing, University of Virginia, Charlottesville, VA, USA
Challenges to the body’s host defense, such as infections, cancer, or inflammation serve as physiological stressors. For the
body to respond appropriately, the brain must be alerted to
the challenge, and initiate appropriate host defense responses. To this end, viscerosensory pathways detect immune- or
pathogen-related signals, and convey this information to stress
response systems in the brain. The viscerosensory pathways
from the body involve both neural and humoral mechanisms.
Of the neural pathways, the vagus is most important for signaling the brain. In addition, spinal nerves signal pain and inflammation in a parallel pathway. Immune and cancer related
signals activate “danger pathways” originating in the caudal
brainstem that drive neuroendocrine (HPA) responses to stress
via their terminations in the PVN. These pathways involve primarily catecholaminergic neurons in the ventrolateral medulla
and rostral nucleus of the solitary tract. A separate pathway
from area postrema targets the parabrachial nucleus of the
pons, which in turn influences other stress-sensitive forebrain
regions including the paraventricular thalamus, the central
amygdala and bed nucleus of the stria terminalis. The medial
prefrontal, anterior cingulate, and insular cortex also contribute to the neural network that responds to different categories
of stressors, such as host defense. Together these brain regions
co-ordinate physiological and behavioral adjustments to homeostatic challenges.
SV.4
CELL LOSS AND IMPAIRED NEUROPLASTICITY
IN PREFRONTAL CORTEX AND HIPPOCAMPUS
IN MAJOR DEPRESSIVE DISORDER (MDD)
Stockmeier C.1, Mahajan G.1, Kooiman H.1, Austin M.1,
Szewczyk B.2, Iyo A.1, May W.1, Rajkowska G.1
1
Psychiatry, University of Mississippi Medical Center, Jackson, MS,
USA; 2 Institute of Pharmacology PAS, Kraków, Poland
Studies in postmortem brain reveal changes in the density and
size of neurons and glia in cerebral cortex and the hippocampus.
Molecular mechanisms may underlie these changes and volume
decreases in depression. In situ hybridization and immunohistochemistry were used to estimate expression of mRNA for tyrosine kinase B (TrkB), length of axons expressing the serotonin
transporter (SERT) and expression of polysialylated neural cell
adhesion molecule (PSA-NCAM) in the hippocampus of subjects with MDD and control subjects. In MDD, there was a significant decrease in expression of mRNA for TrkB, the receptor for brain-derived neurotrophic factor, in CA2/3, and in the
mean total length of axons expressing SERT-immunoreactivity
(IR) in CA1. Area fraction of PSA-NCAM-IR was significantly
increased in the hilus only in MDD with an antidepressant prescription.Decreased expression of TrkB receptor mRNA may
affect cell survival and synaptic plasticity in depression. The
decrease in length of axons expressing the serotonin transporter
suggests a decrease in neuropil or in serotonergic innervation
of CA1 in depression. The role of PSA-NCAM in establishing
synaptic contacts and regulating neurite growth may be increased in subjects treated for depression. Cellular changes at
the microscopic level plus neuroimaging changes detected in
vivo provides an integration of clinical and basic research for
disentangling the pathophysiology of depression.
SYMPOSIUM VI
Genes and Behavior
SVI.1
IT IS SAID THAT “ALMOST MAKES DIFFERENCE”:
ARE DIFFERENT STRAINS OF MICE THE SAME?
Juszczak G.
Department of Animal Behavior, Institute of Genetics and Animal
Breeding, Jastrzębiec, Poland
Both inbred (A/J, AKR, Balb/c, C3H, C57BL/6, DBA/2, FVB) and
outbred (Swiss Webster, CD-1) strains of mice are commonly used
in in vivo and in vitro experiments. Behavioral and pharmacological comparisons of different strains of mice revealed high variability in a number of behavioral parameters and in sensitivity to
pharmacological treatments. Between-strain differences were observed in sensitivity to antidepressant drugs, morphine, MK-801,
kainate and amphetamine. There are both highly sensitive strains
and strains resistant to action of drugs. Less often are observed opposite reactions to pharmacological treatments. The genetic background is, therefore, an important factor contributing to variability
of results obtained in different laboratories using different strains
of mice. This problem is important not only in behavioral pharmacology but also in in vitro experiments and in genetic engineering.
SVI.2
STRESS AND ADAPTATION IN BRATTLEBORO RATS
LACKING ARGININE-VASOPRESSIN
Zelena D.1, Tímár J.2, Bagdy G. 3
1
Behavior and Stress Studies, Institute of Experimental Medicine
HAS, Budapest, Hungary; 2 Department of Pharmacology and
Pharmacotherapy, 3 Department of Pharmacodynamic, Semmelweis
University, Budapest, Hungary
Vasopressin (AVP) has fundamental role in the regulation of
the hypothalamo-pituitary-adrenal axis, the main target of
stress and adaptation. Its role is especially important during
chronic load, which can induce anxiety and depression. The
Symposia 283
importance of AVP in stress and related mood disorders were
studied in spontaneously mutated AVP-deficient Brattleboro
rats (di/di). AVP was confi rmed to be a prominent regulator
of the adrenocorticotrop hormone secretion in wide range of
stresses. Accompanied corticosterone elevation was reduced in
di/di rats just occasionally and remained elevated longer. Endogenous AVP acts as a paracrine signal to facilitate the return
of plasma corticosterone to basal levels. Behavioral studies
confi rmed that di/di animals were less anxious in the defensive
withdrawal test and revealed lower depressive indeces during
forced swimming. Thus, AVP-antagonists are promising targets for drug development.
SVI.3
MEASUREMENT OF PERSISTENTLY EXPRESSED
BEHAVIOURAL TRAITS IN OUTBRED RATS:
UNDERLYING GENE EXPRESSION PATTERNS
Alttoa A., Kõiv K., Harro J.
Department of Psychology, University of Tartu, Tartu, Estonia
Affective disorders are often accompanied by changes in anxiety and motivation to engage in active behaviours. We have
developed an exploration-based behavioural test which allows
distinguishing between rats belonging to clusters with persistently high anxiety/low motivation to explore the environment
and low anxiety/high motivation to explore (LE- and HE-rats,
respectively). The LE-rats are consistently more anxious and
use passive coping strategies in several tests of affective disorders, and have concordant deviations in the monoaminergic
neurochemistry and brain oxidative metabolism. Genome-wide
microarray analysis of gene expression in three brain regions
known to be involved in mood disorders (raphe, hippocampus,
and the frontal cortex) revealed significant alterations in the expression of several neurotransmission-related genes expected to
be relevant to affect (e.g. downregulation of Gabra1, Gabra4,
Gabrg1, Gabrg2, Glrb, Htr1a, Htr2c, Tph2, and upregulation
of Gria3, Grin2a, and Cnr1) in the LE- vs. HE-animals. Also,
overrepresentation of differentially expressed genes involved
in the Wnt signalling pathway in all three brain regions, and
the MAPK signalling, long-term potentiation and long-term
depression pathways in the hippocampus and the frontal cortex suggests that the behavioural differences between rats with
persistently high or low exploratory activity may be related to
differences in synaptic plasticity in the raphe nuclei as well as
its projection areas.
SYMPOSIUM VII
Stem Cells Based Therapeutic Approaches
SVII.1
ARTIFICIAL NEURAL TISSUE FROM CORD BLOOD
FOR TISSUE REPLACEMENT THERAPY OF CENTRAL
NERVOUS SYSTEM
Jurga M., Ali H., Forraz N., McGuckin C.
Department of Neurobiology, Cell Therapy Research Institute,
CTI-Lyon, France
Tissue engineering gives new tools for stem cell-based regenerative medicine and in vitro toxicology. Among different
sources of stem cells human cord and cord blood are safe, clinically and ethically approved and easily obtainable sources of
pluripotent stem cells able to differentiate into many tissues
originated from 3 germ layers. Recently we showed that functional artificial neural tissue can be generated from pluripotent stem cells derived from freshly isolated human cord and
cord blood stem cells in fully-defi ned culture conditions. We
showed that cell starving and precise sequential introduction of
single growth factors and morphogenes resulted in differentiation of pluripotent stem cells towards mature neurons in scaffold-based three-dimensional environment. The pluripotent
stem cells decreased expression of the key pluripotency transcription factors: Oct4A, Sox2, Nanog, c-Myc, and acquired
phenotypes of neuroblasts (Nestin+/GFAP+) followed by generation of mature neuronal networks (NeuN+, PSD95+, TUJ1+,
S100beta). Finally we showed that cell–cell interactions within
the 3D environment of artificial neural tissue were crucial for
functional, electrical and calcium, activity of neural networks
in vitro. We conclude that tissue-engineering approach is important for generation of functional neural tissue in vitro from
cord and cord blood stem cells and therefore might be useful for
future therapies of central nervous system.
SVII.2
FUNCTIONAL NEUROGENIC POTENCIES OF HUMAN
UMBILICAL CORD BLOOD-DERIVED MULTIPOTENT
STEM CELLS THROUGH MULTIPLE MECHANISMS IN
MICE WITH NEURODEGERATIVE DISEASES
Kang K.
College of Veterinary Medicine, Seoul National University, Seoul,
Korea
Stem cell transplantation in neurodegenerative diseases including Alzheimer’s disease has been reported. In this study, we
hypothesized that human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) have the ability to differentiate into functional neurons in the brain. To test this hypothesis,
we verified this assumption in a mouse model of Niemann-Pick
type C (NPC1-/-) disease using hUCB-MSCs. hUCB-MSCs were
transplanted into the hippocampus of asymptomatic mice with
NPC1-/- disease. The transplantation resulted in the recovery of
motor function and dramatically prolonged the survival of the
NPC1-/- mice. Interestingly, many of the transplanted hUCBMSCs showed microtubule associated protein 2-positive cells
with electrophysiological function in the brains of the NPC1/- mice. To examine if the hUCB-MSCs affect endogenous cell
284
survival in the brain, we performed western blotting using mice
brains. These results showed that the hUCB-MSCs can activate
the Phosphoinositide 3-kinase-Akt-glycogen synthase kinase 3
beta ser9 and JAK2-Stat3 pathways for neuronal survival and
regeneration in the brain by secreting functional factors. We
concluded that hUCB-MSCs could contribute to extending the
life of the NPC1-/-mice with the recovery of motor function
by the differentiation into functional active neurons and cell
survival signal cascade. Therefore, it is suggested that hUCBMSCs may be applicable in a broad range of neurodegenerative
diseases.
SVII.3
EMBRYONIC AND ADULT STEM CELLS
FOR TREATMENT OF STROKE
Reymann K.
Department of Neuropharmacology, Leibniz Institute for
Neurobiology, Magdeburg, Germany
Grafted stem cells may confer beneficial effects to the recipient
brain in a twofold manner: (1) they produce a cocktail of trophic
factors and thus promote endogenous repair mechanisms and
also act neuroprotective for endangered neurons and (2) they
differentiate into neurons and glial cells and replace dead cells.
We have transplanted pre-differentiated mouse EGFP+ embryonic stem cells 7 days after experimental stroke into rats.
After 4 weeks cells had been differentiated into neurons and
astrocytes. We detected several sub-types of neurons by means
of neurotransmitter expression and electric activity. However,
cells displayed limited migratory activity and cell survival declined substantially after 12 weeks. Bone marrow stromal cells
(MSC) have been described to be neuroprotective. We found
that MSC are neuroprotective to organotypic hippocampal slice
cultures subjected to an oxygen-glucose deprivation (OGD)
even without having direct cell–cell contact. This effect was
increased if MSC were treated by complete serum deprivation before delivery to OGD treated slices. Interestingly, OGD
damaged slices stimulated the MSC to produce certain growth
factors like bFGF and NGF. A complete serum-deprivation induces the generation of a subpopulation of small-sized cells expressing nestin, GFAP, Sox-2 and Oct4 and by this resembling
both neural and embryonic stem stem cells. It remains open
whether this sub-population has the potential to differentiate
into neuroectodermal cells.
SYMPOSIUM VIII
Cholinergic System in Neurodegereneration
SVIII.1
AMYLOID PRECURSOR PROTEIN AND
CHOLINESTERASES IN BRAIN FUNCTION
AND NEURODEGENERATION
Nalivaeva N.
Institute of Molecular and Cellular Biology, University of Leeds,
Leeds, UK
Amyloid precursor protein (APP) and acetylcholinesterase (AChE)
are membrane-bound proteins possessing a variety of roles in brain
development and functioning. While APP is mostly known as a protein producing amyloid β peptide (Aβ) both AChE and butyrylcholinesterase (BuChE) are generally linked to cholinergic neurotransmission. All three proteins have soluble forms which, despite their
different origin, are believed to play a role in neurogenesis, synaptogenesis and neuroprotection. There is evidence that APP and AChE
can form complexes in the membrane and both AChE and BuChE
are found in tight association with Aβ in senile plaques characteristic for Alzheimer’s disease. Under certain circumstances AChE is
shed from the neuronal membrane by an enzymatic process similar
to that involved in the non-amyloidogenic shedding of APP. Our data
demonstrated that the content of membrane bound and soluble forms
of APP, AChE and BuChE significantly increase in various brain
structures of rats during the first month of postnatal life suggesting
their role in brain development and synaptogenesis. The levels of
soluble and membrane-bound forms of these proteins were affected
by prenatal hypoxia with subsequent developmental deficit observed
in these animals. The molecular mechanisms of the APP and AChE
shedding and their involvement in the pathogenesis of AD will also
be discussed. Supported: the UK MRC, RAS “Fundamental sciences
to medicine”.
SVIII.2
CHOLINOTOXICITY OF β-AMYLOID
Schliebs R.
Paul Flechsig Institute for Brain Research, University of Leipzig,
Leipzig, Germany
Many Alzheimer patients demonstrate also cerebral amyloid angiopathy and microvascular degeneration. The upregulation of the
angiogenic vascular endothelial growth factor (VEGF) in brains
of Alzheimer patients in close relationship to β-amyloid plaques,
suggests a link of VEGF action and processing of the amyloid
precursor protein (APP). As the brain vascular system underlies
a functional control by basal forebrain cholinergic terminals, the
question arises of whether there is a relationship between damage
of brain capillaries, VEGF upregulation, and cortical cholinergic
denervation. Tg2576 mice, that express the Swedish double mutation of human amyloid precursor protein and progressively develop
β-amyloid deposits, were used to study age- and β-amyloid related
changes in cerebral cortical microvessels, VEGF level, and associated cholinergic terminals. Treatment of brain slices with VEGF affected the formation of soluble β-amyloid, while the overproduction
of β-amyloid led to significant decrease of cholinergic fibre density
in cortical regions of aged transgenic mice. The data further sug-
Symposia 285
gest that changes in cholinergic innervation of microvessels may
contribute to pathological alterations of the cerebrovascular system.
Supported by IZKF, University Leipzig, and Alzheimer Forschungsinitiative (AFI).
SVIII.3
PROTEOLYTIC CASCADES IN THE BRAIN
AND THEIR CHOLINERGIC REGULATION:
ROLE IN NEURODEGENERATION
Turner A.
Institute of Molecular and Cellular Biology, University of Leeds,
Leeds, UK
Progressive dysfunction of cholinergic neurotransmission in selected
brain regions contributes to the cognitive and behavioural disturbances in Alzheimer’s disease although the underlying mechanisms
remain unclear. The amyloid β-peptide (Aβ) is a major player in Alzheimer progression and its levels are modulated by the cholinergic
system. The level of Aβ in the brain represents a dynamic equilibrium state as a result of its biosynthesis from the amyloid precursor protein (APP) by β- and γ-secretases, its alternative processing
by α-secretases and its degradation by a team of amyloid-degrading
enzymes. The first and rate-limiting step in amyloid synthesis, mediated by the membrane-bound β-secretase occurs in lipid raft domains
and is modulated by other interacting proteins, e.g. the prion protein.
Alternatively, and by far the major metabolic route for APP, is its processing in non-raft domains, to release the soluble extracellular domain, sAPPα, in a process mediated by a cohort of ADAMs zinc metalloproteases and regulated through cholinergic signalling pathways.
We have used a variety of biochemical and genetic techniques to identify the proteases involved in these processes, their regulation and
their subcellular localizations. Manipulation of cholinergic signalling
could provide one strategy for reducing amyloid burden alongside direct targeting of the amyloid-forming and -degrading enzymes. This
work was supported by the UK Medical Research Council.
SVIII.4
PHENOTYPE AND ENERGY DEPENDENT
CHOLINOTOXICITY
Szutowicz A., Bielarczyk H., Ronowska A., Dys A., Gul-Hinc S.,
Jankowska-Kulawy A.
Department of Laboratory Medicine, Medical University of Gdansk,
Gdańsk, Poland
Cognitive deficits in Alzheimer’s disease (AD) are caused by preferential loss of septal cholinergic neurons. We postulate that utilization of acetyl-CoA for ACh synthesis creates its deficit for energy production in cholinergic neurons. Therefore, we investigated
whether higher expression of the cholinergic phenotype could
make these neurons more susceptible to putative AD pathogens.
Chronic exposure of nondifferentiated (NC) and differentiated
(DC) cholinergic septal neuroblastoma SN56 cells to amyloid-β,
NO excess and Al inhibited pyruvate dehydrogenase (PDH) and
other oxidative enzymes activities in, with similar potencies. However, they caused much greater decrease of acetyl-CoA content,
cholinergic functions and viability in DC than in NC, respectively.
On the other hand, both in NC and DC, Zn caused acute inhibition
of PDH, aconitase and NADP isocitrate dehydrogenase, with similar Ki values of about 0.058, 0.010 and 0.005 mM, respectively. Un-
expectedly, Zn-Ki for ketoglutarate dehydrogenase (KDH) in DC
was 0.0005 mM, whereas in NC 0.0040 mM, respectively. Significant correlations were found between acetyl-CoA in mitochondria
and mortality as well as between cytoplasmic acetyl-CoA and expression of the cholinergic phenotype in SN56 cells. We postulate
that these two partially independent pools of acetyl-CoA affect
survival and transmitter functions of cholinergic neurons, respectively. Supported by MNiSW grants 2P05A 11030, NN401139933
and AMG project St-57.
SYMPOSIUM IX
Protein Toxicity and Molecular Mechanisms
of Neurodegenerative Diseases
SIX.1
REGULATION OF NEUROTRANSMITTER SECRETION
BY SPHINGOSINE-1-PHOSPHATE
Nakamura S., Okada T.
Department of Biochemistry, Kobe University Graduate School of
Medicine, Kobe, Japan
Neuronal activity greatly influences the formation and stabilization
of synapses. Although receptors for sphingosine-1-phosphate (S1P),
a lipid mediator regulating diverse cellular processes, are abundant
in CNS, neuron-specific functions of S1P remain largely undefined.
Here we present novel actions of S1P using primary hippocampal
neurons as a model system, i.e. S1P triggers neurotransmitter secretion in a depolarization-independent manner. Sphingosine kinase 1
(SK1), a key enzyme for S1P production, was enriched in functional
puncta of hippocampal neurons. Silencing SK1 expression by siRNA resulted in strong inhibition of depolarization-evoked glutamate
secretion. FRET analysis demonstrated that S1P1 receptor at the
puncta was activated during depolarization and that depolarizationinduced S1P1 receptor activation was inhibited in SK1-knockdown
cells. Importantly, exogenously added S1P at nanomolar concentration by itself elicited glutamate secretion from hippocampal cells
even when Na+-channel was blocked by tetrodotoxin, suggesting
that S1P acts on presynaptic membranes. These findings indicate
that S1P, through its autocrine action, facilitates spontaneous glutamate secretion. We are now confirming these findings using electrophysiological approaches and also studying the role of S1P in
memory formation and learning using knockout mice.
SIX.2
HIV-1 AND AMYLOID BETA PEPTIDE INTERACTIONS
AT THE BLOOD–BRAIN BARRIER LEVEL
Toborek M., András I.
Molecular Neuroscience and Vascular Biology Laboratory,
Department of Neurosurgery, University of Kentucky, Lexington,
KY, USA
An increase in the older population infected with HIV-1 is an emerging development in HIV-1 epidemiology. Aging is connected with
increased deposition of amyloid beta peptide (amyloid beta) in the
brain. In the current study, we propose that amyloid beta and HIV-1
can potentiate their toxic effects at the blood–brain barrier (BBB)
286
level. To address this notion, we employed an in vitro model of human brain microvascular endothelial cells (HBMEC) directly exposed to HIV-1 or co-cultured with HIV-1 infected human monocytes. Exposure of HBMEC to amyloid beta (1-40) in the presence of
HIV-1 resulted in a markedly increased amyloid beta binding/entry
into HBMEC. We then hypothesized that HIV-1 may either increase
binding/entry of externally added amyloid beta or elevate the amount
of endogenously produced amyloid beta. The receptor for advanced
glycation end products (RAGE) is known to be involved in the transport of amyloid beta across the BBB into the brain. RAGE immunoreactivity was stronger and RAGE protein levels were elevated in
HBMEC exposed to HIV-1 as compared to control. In contrast, exposure to HIV-1 decreased expression of lipoprotein receptor related
protein-1 (LRP1) which is the main receptor that transports amyloid
beta from the brain to blood. These results indicate that HIV-1 can
decrease the ability of the BBB to transport amyloid beta from the
brain and thus predispose the brain to increased amyloid beta accumulation. Supported by MH072567, MH63022, and NS39254.
SIX.3
ALPHA SYNUCLEIN AND ITS NEUROTOXIC
FRAGMENT NAC IN NEURODEGENERATION
Adamczyk A., Kazmierczak A., Strosznajder J.
Department of Cellular Signaling, Mossakowski Medical
Research Centre PAS, Warszawa, Poland
accumulation of Aβ peptide is a critical event in the early pathogenesis of AD. This lecture will review key steps of Aβ metabolism
and their disturbances leading to build-up of Aβ in the AD brain.
Development of therapeutic approaches targeting Aβ cascade will
be subsequently discussed. They include passive and active immunization against Aβ, inhibitors of β and γ secretases, RAGE inhibitors, β-sheet breakers and approaches targeting the apolipoprotein
E (apoE). ApoE is a critical factor promoting Aβ deposition in the
brain and affecting its clearance. The magnitude of the apoE/Aβ interaction is isoform specific, providing an explanation for the linkage between the apoE4 allele and an increased risk of sporadic AD.
Our laboratory has demonstrated that blocking the apoE/Aβ binding
with synthetic peptide-Aβ12-28P, which mimics the apoE binding
site on Aβ, reduces the burden of vascular and parenchymal Aβ deposits in AD transgenic mice and prevents them from developing a
memory deficit. Ongoing research pursues development of peptidomimetic derivatives of Aβ12-28P with improved therapeutic efficacy
and biostability with the aim to obtain a lead therapeutic compound
for clinical investigations. Support: Dorothy D. Eweson Lectureship
on the Advances in Aging Research, grants AG24847, AG31221.
SYMPOSIUM X
Stroke-Induced Brain Reorganization
Alfa-Synuclein (ASN), a small (14 kDa) presynaptic protein abundant in the brain play significant role in pathogenesis of Parkinson’s
disease (PD) and is implicated in the other neurodegenerative disorders. The central domain of ASN, the non-amyloid β component
of Alzheimer’s disease amyloid (NAC) could be responsible for
its toxicity. Our study showed the significant role of ASN/NAC in
disturbance of dopamine transporter (DAT) and voltage-dependent
calcium channels (VDCC) function in the brain. ASN-evoked disregulation of Ca2+ homeostasis leads to activation of neuronal nitric
oxide synthase (nNOS). Our data indicated NO-dependent modification of several proteins involved in decision of cell life/death
including caspase-3 and poly(ADP-ribose) polymerase 1 (PARP-1).
The further study carried out on cells in culture presented ASN/
NO dependent mitochondria failure and indicated that NO pool liberated by ASN activates caspase-3 that leads to PARP-1 degradation. Inhibitor of NOS (NNLA), caspase-3 (Z-DEVD-FMK) and a
mitochondrial permeability transition pore blocker, cyclosporine A
protected cells against ASN evoked cell death. Our results indicate
that ASN/NAC leads to NO mediated mitochondria dysfunction
and caspase-dependent programmed cell death. Supported by the
Ministry of Science and Higher Education Grant 2PO5A4129 and
Scientific Network 28/E-32/BWSN-0053/2008.
SX.1
EXPLORING THE CHANCES OF STEM CELL BASED
REGENERATION AFTER STROKE BY MRI
Hoehn M.
In-vivo-NMR-Laboratory, Max Planck Institute for Neurological
Research, Köln, Germany
SIX.4
THERAPEUTIC APPROACHES TARGETING
Aβ CASCADE IN ALZHEIMER’S DISEASE
Sadowski M.
Departments of Neurology, Pharmacology, and Psychiatry, New York
University School of Medicine, New York, NY, USA
SX.2
HOW PHOTOTHROMBOTIC STROKE AFFECTS
EXTRACELLULAR MATRIX?
Nowicka D., Karetko M., Aleksy M., Skangiel-Kramska J.
Department of Molecular and Cellular Neuroscience, Nencki Institute
Alzheimer’s disease (AD) is the most common cause of dementia,
affecting nearly five million patients in the USA and 20 million
worldwide. The β-amyloid (Aβ) cascade hypothesis maintains that
Perineuronal net (PN), a lattice-like structure surrounding certain neurons, represents a special form of extracellular matrix
of the brain. The role of this structure remains elusive. It has
Stem cell based regeneration after stroke is a concept of investigating the chronic phase, following stroke and stem cell implantation.
Therapeutic success is measured assessing functional deficit and
improvement with functional brain activation studies by fMRI. We
demonstrate an animal fMRI protocol to exclude spontaneous recovery as a confounding factor of therapeutic improvements. Stem
cell implantation into the ischemic hemisphere of those animals
without spontaneous recovery shows functional improvement beginning approximately ten weeks after implantation. This functional improvement is confirmed in electrophysiological recordings of
somatosensory evoked potentials and supported by sensorymotor
behavioral test results. Further experiments on transgenic mice allow to monitor neurogenesis of new cells migrating from the subventricular zone to the ischemic territory. For this new approach, a
multimodal molecular imaging approach is used, combining optical
and magnetic resonance imaging techniques in vivo.
Symposia 287
been suggested to regulate neuronal plasticity, accumulate
trophic molecules and protect neurons from damaging factors.
Here we studied whether PN-enwrapped neurons are protected against ischemic injury. To this aim we performed cortical
photothrombotic stroke in rats and investigated PNs and their
co-localization with Fluoro Jade C (FJ), a marker of rapid cell
death. We found that in the ischemic core 4 h after the stroke
still PNs were observable, none of them around FJ-positive neurons. No PNs were detected after 24 h. In the peri-infarct area,
a huge loss of PNs was observed 24 h after stroke, followed by a
partial restoration 30 days post-infarct. Similar effect was found
in remote cortical areas, except that in this case the restoration
was almost complete already 7 days after stroke. No FJ staining was observed outside ischemic core. These results suggest
that in areas that are not directly damaged, ischemic insult disrupts PNs but the neurons survive this disruption. Supported by
MNiSW Grant N3030300832/0474.
SX.3
fMRI STUDY OF RECOVERY FROM UNILATERAL
NEGLECT
Seniow J1., Polanowska K.1, Marchewka A.2, Jednoróg K.2,
Członkowska A.1,3, Grabowska A.2, Królicki L. 3, Kossut M.2
1
Institute of Psychiatry and Neurology, Warszawa, Poland; 2Nencki
Institute of Experimental Biology PAS, Warszawa, Poland; 3Medical
University of Warsaw, Warszawa, Poland
Abstract not received
SX.4
BRAIN PLASTICITY FOLLOWING STROKE: POSSIBLE
TARGETS FOR THERAPEUTIC INTERVENTION
Witte O.
Department of Neurology, Friedrich-Schiller-University, Jena,
Germany
Abstract not received
SYMPOSIUM XI
Glutamatergic Receptors as a Target
for Future Psychotropic Drugs Action
SXI.1
DIFFERENTIAL ROLES OF mGluR2 AND mGluR3
IN THE BRAIN
Wroblewska B.
Georgetown University, Washington DC, USA
Group II metabotropic glutamate receptors (mGluR2 and mGluR3)
are negatively coupled to adenylate cyclase and cAMP transduction
system. They decrease cAMP through a pertussis toxin-sensitive Gprotein coupled mechanism. Recently we have shown that mGluR3
expressed in rat cerebellar granule cells and astrocytes also may be
coupled through a G-protein mechanism to a cGMP transduction
pathway. To test the hypothesis that mGluR2 and mGluR3 share a
coupling to the cGMP pathway, their cDNA were transfected into C6
glioma cells, characterized by robust cGMP response, and their coupling to cAMP and cGMP transduction pathways was characterized.
Consistent with data from neurons and astrocytes, mGluR3 stably
expressed in C6 cells responded to agonists with reductions in cGMP
levels. In contrast, the mGluR2 receptor was negatively coupled to
cAMP in C6 cells but not to cGMP, indicating the functional difference between these two receptors. The coupling to cGMP signal transduction depends on the cell type – it is pertussis toxin-sensitive in the
astrocytes and C6 mGluR3 cell line, while it is not in the cerebellar
granule cells. In all investigated cell types the coupling was not mediated by the interactions with phosphodiesterase (IBMX-insensitive).
Evidence that human mGluR3 (GRM3 gene) may be a susceptibility gene for schizophrenia and prospective therapeutic potential of
group II agonists may speed up the development of selective agonists
or positive allosteric modulators for these two receptors
SXI.2
SELECTED ASPECTS OF GROUP I mGlu RECEPTOR
NEGATIVE AND POSITIVE MODULATORS
Danysz W.
Department of In Vivo Pharmacology, Merz Pharmaceuticals,
Frankfurt am Main, Germany
Several mGluR5 modulators have became available showing acceptable pharmacokinetics and selectivity. There are negative allosteric
modulators such as MTEP, MPEP, or fenobam (NAMs) or positive
allosteric modulators (PAMs) such as CDPPB or ADX47273. We
performed a verification of the therapeutic potential of these modulators using various behavioural methods. mGluR5 NAMs produced
analgesic effects in several pain models such as formalin, and Freund
adjuvant model of inflammatory pain. Additionally they very strongly
attenuated L-DOPA induced dyskinesia and produced anxiolytic activity in some models of anxiety such as fear potentiated startle, context
freezing. However, efficacy was not better then respective indication
reference agents. mGluR5 PAMs are expected to have antipsychoticlike activity and improve learning. These expectations can be in our
hands only partially fulfilled for schizophrenia (positive effects in amphetamine-induced hyperactvitivity, apomorphine-induced prepulse
inhibition, or conditioned avoidance response inhibition). No clear
positive effect was observed in models of cognition. Recently, several
clinical trials with metabotropic glutamate receptor type 5 negative
288
modulators showed positive effects in indications such as migraine, reflux and L-DOPA-induced dyskinesia. This regards ADX 10059 from
Addex and AFQ056 from Novartis indicating that their introduction to
clinical practice may be just a matter of short time.
SXI.3
CAN LIGANDS OF METABOTROPIC GLUTAMATE
RECEPTORS BECOME ANTIPARKINSONIAN DRUGS?
Ossowska K.
Department of Neuro-Psychopharmacology, Institute of
Pharmacology PAS, Kraków, Poland
Symptoms of Parkinson's disease (akinesia, muscle rigidity and tremor) result from degeneration of dopaminergic nigrostriatal pathway.
Furthermore, a shift of dopaminergic-glutamatergic balance towards
overactivation of glutamatergic systems exists in this disease. Therefore, drugs which inhibit glutamatergic transmission were expected to
have antiparkinsonian impact. Our studies showed that the blockade
of the group I mGluRs (mGluR1 and 5) in the striatum decreased
catalepsy and muscle rigidity in the haloperidol-induced animal models of parkinsonism, which seemed to be due to inhibition of the indirect striopallidal pathway. On the contrary, systemic injections of
mGluR5 antagonists did not influence the tacrine-induced tremor and
diminished functioning of the nigrostriatal pathway. Moreover, the
mGluR1 antagonist strongly enhanced the tremor induced by harmaline. Agonists of groups II and III mGluRs exerted antiparkinsonianlike effects which differed depending on their brain targets. Agonists
of the group II seemed to act at the level of the subthalamo-nigral
glutamatergic synapses, but not in the striatum, whereas an antiakinetic effect of agonists of group III was strong in the globus pallidus
and striatum, but weak or none in the substantia nigra. The above
results seem to suggest that because of a wide distribution of mGluRs
in the brain, ligands of these receptors may exhibit limited efficiency
as antiparkinsonian drugs in humans.
SXI.4
METABOTROPIC GLUTAMATE RECEPTOR LIGANDS
AS A POSSIBLE TARGETS FOR TREATMENT OF
ANXIETY AND DEPRESSION
Pilc A., Branski P., Palucha-Poniewiera A., Stachowicz K.,
Wieronska J.
Department of Neurobiology, Institute of Pharmacology PAS,
Kraków, Poland
Preclinical data indicated that antagonists of group I mGlu receptors, particularly antagonists of mGlu5 receptors, produced both
anxiolytic-like and antidepressant-like effects. Clinical data also
demonstrated that mGlu5 receptor antagonist, fenobam, was an active anxiolytic drug. The anxiolytic effects exerted by mGlu5 receptor antagonists are profound, comparable to or stronger than those of
benzodiazepines. Among all mGlu receptor ligands, group II mGlu
receptor agonists seem to be drugs with promising therapeutic potential and good safety profile. Animal studies showed anxiolytic-like
effects of group II mGlu receptor agonists. Currently, group II mGlu
receptor agonists are in phase III clinical trials for potential treatment
of anxiety disorders. On the other hand, data have been accumulated
indicating that antagonists of group II mGlu receptors have an antidepressant potential. Group III mGlu receptor ligands represent the least
investigated group of mGlu receptors. The preclinical data, however,
indicate that a selective agonist of mGlu4 and Glu8 receptors- ACPT-I
produced anxiolytic but not antidepressant effects after peripheral administration, while the selective positive allosteric modulator of Glu7
receptors, AMN082 produced both anxiolytic and antidepressantlike. The data show that the effects of group III mGlu receptor ligands
may differ dependently of their receptor subtype specificity
SYMPOSIUM XII
Interaction Between Antidepressant Drugs
and Immunoendocrine Systems
SXII.1
MICE WITH ALTERED GLUCOCORTICOID RECEPTOR
EXPRESSION AS A MODEL FOR DEPRESSION
Gass P.
Central Insitute of Mental Health, Mannheim, Germany
Impaired glucocorticoid receptor (GR) signaling is a postulated
mechanism for the pathogenesis of major depression. Since in vivo
expression and functional studies of GR are not feasible in humans,
we have generated mouse strains that over- or underexpress GR: (1)
GR heterozygous mice (GR+/-) with a 50% GR gene dose reduction;
and (2) GR transgenic mice (YGR) with a 100% gene dose elevation. GR+/- mice exhibit normal baseline behaviors, but demonstrate
after stress exposure increased helplessness, a behavioral correlate
of depression in mice. Similar to depressed patients, GR+/- mice have
a disinhibited HPA system and a pathological DEX/CRH test. Thus,
they represent a murine depression model with good face and construct validity. YGR mice, in contrast, show reduced helplessness
after stress exposure, and an improved HPA system feedback regulation. Therefore they are a model for a stress-resistant strain. These
models can be used to study plasticity changes underlying the pathogenesis of depressive disorders. As first potential molecular correlate we identified a downregulation of BDNF in the hippocampus
of GR+/- mice. Translational approaches, i.e. how to use these models
specifically for clinically relevant questions, will be discussed.
SXII.2
EFFECTS OF ANTIDEPRESSANTS ON CYTOKINES
IN EXPERIMENTAL STUDIES
Obuchowicz E.
Department of Pharmacology, Medical University of Silesia,
Katowice, Poland
Cytokines play an important role as immunotransmitters and coordinate the activity of the endocrine, immune and neurotransmitter
systems. The altered cytokine plasma levels have been found in
some groups of depressed patients. Administration of proinflammatory cytokines (IL-1β, TNF-α) or unspecific stimulator of their
synthesis – lipopolysaccharide produces in lab animals “sickness
and depressive-like behavior”. These behavioral effects are attenuated by pretreatment with some antidepressants. Results of the investigation carried out on transgenic mice support suggestion that
proinflammatory cytokines may be involved in pathogenesis of depressive symptoms. In majority of in vivo and ex vivo studies it has
been observed that chronic administration of some antidepressants
shifts peripheral balance between IL-1β, TNF-α and antiinflam-
Symposia 289
matory cytokine – IL-10 towards the latter. Antiinflammatory and
neuroprotective effects of various antidepressive drugs were confirmed using cultured brain cells. Data from these in vitro studies
indicate that antidepressants suppress IL-1β, TNF-α gene expression likely due to blockade of NFκ-B and p38 MAPK pathways. It
is suggested that changes in cytokine networks might be of significance for antidepressive and/or analgesic action of antidepressants
and may justify their use in therapy of neurological diseases accompanied by elevated levels of proinflammatory cytokines.
SXII.3
NEUROPLASTICITY IN DEPRESSION
Almeida O.1, Cerqueira J.2, Roselli F.1, Bessa J.2, Sousa N.2
1
NeuroAdaptations Group, Max Planck Institute of Psychiatry,
Munich, Germany; 2 Department of Neuroscience, Institute of Life
& Health Sciences, University of Minho, Braga, Portugal
Depression may be viewed as a disorder of networks that interconnect different brain areas to which predominant roles in the regulation of individual behavioural domains (mood, anxiety, cognition
and reward) that contribute to the depressive state have been ascribed. Using an animal model of depression (chronic unpredictable
stress) as well as primary neuronal cell cultures, our studies have
focused on volumetric and cell number changes (including those
resulting from neurogenesis and apoptosis) in the prefrontal cortex
and hippocampus, as well as on patterns of dendritic arborization
and signs of synaptic plasticity that parallel the manifestation of
depressive-like behaviours. They have also begun to cast light on
some of the cellular mechanisms underlying these changes; in this
respect, insights into the requirements for maintaining synaptic
structural and functional integrity have been gained from work
with Alzheimer’s disease-related proteins and peptides. Besides
identifying potential targets for antidepressant drugs, our studies
add support to the network hypothesis of depression.
SXII.4
THE EFFECT OF ANTIDEPRESSANT DRUGS ON
IMMUNOENDOCRINE CHANGES IN ANIMAL MODELS
OF DEPRESSION
Budziszewska B., Kubera M., Szymanska M., Basta-Kaim A.,
Grygier B., Lason W.
Department of Experimental Neuroendocrinology, Institute of
Exacerbated glucocorticoids and cytokines action are essential
factor in the pathogenesis of depression, and the effects of antidepressant drugs on these parameters are poorly recognized.We
investigated the effect of antidepressant drugs on the HPA axis activity in prenatally stressed S-D rats and on cell-mediated immunity in Wistar rats and C57BL/6 mice subjected to chronic mild
stress (CMS) model of depression. The activity of HPA axis was
estimated by measuring the level of glucocorticoid receptors (GR)
and activity of some kinases which are known to influence GR
action. Adult rats subjected to prenatal stress displayed prolonged
immobility in the Porsolt test and in open-field test, elevated corticosterone level, increased GR level in the hippocampus but not in
frontal cortex. They also showed decreased FKBP51 in the frontal cortex, but not in hippocampus, decreased the active, phosphorylated form of the JNK1 and 2 kinase in the hippocampus
and the active form of p38-MAPK in the frontal cortex. Chronic
imipramine, fluoxetine, mirtazapine or tianeptine administration
normalized most of these parameters. In CMS model of depression anti-anhedonic effect of imipramine was accompanied by
decreased proliferative activity of splenocytes and their ability
to produce pro-inflammatory cytokines in rats. In desipramine
treated mice subjected to CMS increased ability of T cells to produce negative immunoregulator IL-10 and decreased the cytotoxic
activity of NK cells were observed.
SYMPOSIUM XIII
Motor Units and Motoneurons
SXIII.1
PHYSIOLOGICAL ADAPTATIONS IN THE RHYTHMIC
FIRING PROPERTIES OF ALPHA-MOTONEURONES
TO CHANGES IN PHYSICAL ACTIVITY AND AGING
Gardiner P.
Spinal Cord Research Center, University of Manitoba, Winnipeg,
Canada
Our previous research has revealed that hindlimb α-motoneurones
respond to increases and decreases in the activity of the organism,
as well as aging, by alterations in biophysical properties measured
in anesthetized rats using intracellular recording techniques. Properties that change include resting membrane potential (RMP), voltage threshold (Vth), afterhyperpolarization (AHP) amplitude, rates
of antidromic spike development, and rheobase. Rhythmic firing
properties that also show changes include minimum and maximum
firing frequencies, frequency/current slopes, and the degree of late
adaptation. Thus, generally speaking, “excitability” of motoneurones
increases and decreases in various models of increased and decreased
chronic activity, respectively, and decreases during aging, with implications for motor unit recruitment during voluntary movement. Single-cell modelling of ionic currents has suggested that these chronic
adaptations most likely involve changes in the transient sodium and
DR potassium ion channels, and perhaps channels associated with the
AHP as well as leak currents. Results from this research program have
implications for treating the neuromuscular system compromised by
disease, trauma, age, and even “chronic sedentarism”.
SXIII.2
MOTOR UNITS ACTIVITY IN EMG, MMG AND FORCE
RIPPLE: THREE PICTURES FOR ONE SUBJECT
Orizio C., Gobbo M., De Grandis D., Ravenni R.
Department of Biomedical Science and Biotechnology, University of
Brescia, Brescia, Italy
Mechanomyogram (MMG) detects the muscle surface oscillations
due to the mechanical activity of the motor units (MU). Even during
static contractions MMG presents macro components (due to the
gross dimensional changes of the muscle-tendon unit from rest to
activity) and micro components (the summation of the dimensional
changes of the fibres of each recruited motor unit). The single MU
mechanical contribution to MMG (MUMC) was defined by Gordon
and Holbourn a the ‘‘mechanical counterpart” of the MU action
290
potential. During voluntary contraction MUMC and MU twitch
(MUT) can be extracted from MMG and force ripple signal by
means of the EMG driven spike triggered averaging technique. In
small hand muscles during 5% of the maximal voluntary isometric
contraction (MVC) the values of MUMCs and MUTs were around
11 mm/s2 and 7 mN, respectively. Already at these low effort levels
the summation in MMG of single MUMCs of the recruited MUs is
not linear. This may be due to the long duration of the MUMCs and
to the changes in the muscle transverse and longitudinal compliance to mechanical stress determined by the overall MUs activity.
On this basis the meaning of the MMG and force ripple oscillations
(when larger of the peak to peak noise of the signals during rest and
of the MUCMs and MUTs reported for single MUs) with respect
to the MUs driving program has to be deeply investigated. This
could be done by a cross analysis with the EMG which may partly
disclose the central nervous system MUs activation strategy.
SXIII.3
CONTRACTIONS OF MOTOR UNITS EVOKED
BY STIMULATION WITH PULSES AT VARIABLE
INTERPULSE INTERVALS
Krutki P.
Department of Neurobiology, University School of Physical Education
in Poznan, Poznań, Poland
The rate of motoneuronal firing is a major factor regulating the force
of motor units (MUs). During voluntary activity of a muscle, its MUs
generate tetanic contractions which are characterized by variable
force and fusion degree. This study aimed at analysis of force changes
during tetani evoked at random stimulation patterns and their mathematical decomposition into responses to individual pulses. It was
demonstrated that longer interpulse intervals and lower initial levels of
force result in higher force increase during next components of tetanic
contractions, and that random stimulation pattern produces higher
output of MUs (and higher economy of contraction) than the constant
frequency during relatively weak contractions. The decomposition
revealed considerable variability in twitch responses to successive
pulses. Their basic parameters can be predicted with high accuracy on
the basis of the force level at which the next contraction begins. The
physiological significance of successive action potentials generated by
active motoneurones appears to show considerable variation, so the
application of constant frequency patterns during experimental analysis of functionally isolated MUs can lead to several conclusions that
do not correspond to activity of MUs during natural voluntary movements. This concerns parameters of successive components of tetanic
contractions: the contraction time, the instantaneous force and its variability, the effectiveness and economy of the MU’s contraction.
SXIII.4
VARIABILITY AND PLASTICITY OF MOTOR UNIT
CONTRACTILE PROPERTIES
Celichowski J.
The motor unit contractile properties respond to changes in activity
and in motor innervation of muscles. The contractions of isolated motor units evoked by the electrical stimulation of very thin filaments of
the ventral root were studied in anesthetized animals. Changes in the
distribution of the three basic physiological types of motor units (fast
fatigable, fast resistant and slow) in the rat medial gastrocnemius muscle and plasticity of their contractile properties as: contraction and relaxation time, force and fatigability were studied in ageing process, as
effects of total and partial spinal cord injury, locomotor and vibration
training. Moreover, distribution and variability of motor unit properties as well as innervation of skeletal muscles in male and female
muscles were investigated. The comparison of motor unit properties
in rat and cat muscle revealed considerable differences in twitch time
and force parameters, distribution of motor unit types in muscles of
the two species whereas the fatigability, ability to potentiate the force,
sag and tetanic phenomenon revealed numerous similarities.
SYMPOSIUM XIV
Advanced Methods of Brain Signal Analysis
SXIV.1
ESTIMATION OF THE TIME-VARYING CORTICAL
CONNECTIVITY CHANGES DURING THE ATTEMPT
OF FOOT MOVEMENTS BY SPINAL CORD INJURED
AND HEALTHY SUBJECTS
De Vico Fallani F.
Department of Physiology and Pharmacology, University of Rome
“Sapienza”, Rome, Italy
In this study we estimated time-varying cortical connectivity patterns from a group of Spinal Cord Injured (SCI) patients during
the attempt to move a paralyzed limb. These data were compared
with the time-varying connectivity patterns estimated in a control
group during the real execution of the movement by using timevarying Partial Directed Coherence. Connectivity was estimated
from high resolution EEG recordings with the use of realistic head
modelling and the linear inverse estimation of the cortical activity
in a series of Regions of Interest of the cortex (ROIs). The experimental evidences obtained support the conclusion that the SCI
population involved a larger cortical network than those generated
by the healthy subjects during the task performance. Such network
differs for the involvement of the parietal cortices, which increases in strength near to the movement imagination onset for the SCI
when compared to the normal population. Such details about the
temporal evolution of the connectivity patterns cannot be obtained
with the application of the standard estimators of connectivity.
SXIV.2
BEYOND THE PSTH: POINT PROCESS MODELING
OF SPIKE TRAINS
Wojcik D., Mochol G., Wypych M., Waleszczyk W., Wrobel A.
A prerequisite for a quantitative theory of neural coding is adequate
description of spike trains. Fifty years ago it was understood that the
probability to generate a spike at a given time from the stimulus onset
– the post-stimulus time histogram (PSTH) – brings in useful information adding to the mean number of spikes in the trial. Today there is a
growing consensus that one must go beyond the PSTH building more
complex point process models of neural activity which can account,
Poster Session I 291
for basic physiological properties of spike firing, e.g. for the refractory
properties or for adaptation mechanisms of the cell. We shall present
some basic concepts of the point process theory in the context of the
spike trains and present a simple method of estimation of a class of
second order processes for stimulus-evoked activity. We will illustrate
the results with an analysis of sample data from the cat superior colliculus. Supported by grants N401 146 31/3239 and 46/N-COST/2007/0.
SXIV.3
BRAIN STIMULATION AND EPILEPSY: PROBING THE
AUTONOMY OF THE NEURAL SYSTEM
Kalitzin S.1, Velis D.2, Lopes da Silva F.3
1
Medical Physics Department, 2 Department of Clinical
Neurophysiology and EMU, Epilepsy Institute of The Netherlands
(SEIN), Heemstede, The Netherlands; 3 Center of Neuroscience,
Swammerdam Institute for Life Sciences, University of Amsterdam,
Amsterdam, The Netherlands
A decreased ability of the brain to retain sufficiently its autonomous
dynamics during stimulation can be indicative for pathological neurological conditions, such as epilepsy. Sensory as well as direct brain
stimulation can be therefore valuable supplementary tools for identification of suspected epileptic disorders and even for localization of
epileptic tissue in the brain. While most stimulation-based approaches rely on spectral or amplitude analysis we argue that important information is contained in the phase clustering index (PCI) derived from
the evoked responses to relatively high-frequencies stimulation (>10
Hz) . We present examples where visual sensitivity can be detected by
phase-related features of the visual evoked potentials. We also present
analysis from intracranial recordings and direct electric stimulation in
patients undergoing pre-operative video/EEG. Our results support the
hypothesis that the patient’s brain occupies states of increased probability of an epileptic transition prior to seizure. Detection of these
states can be relevant as an early warning paradigm. We were also able
to find a correlate between the same features of the electrically evoked
responses and the location of the seizure onset in cases of localization
related epilepsies. We argue that stimulation allows revealing features
that are sometimes undetectable by passive observation techniques.
SXIV.4
MULTICHANNEL DATA ANALYSIS – INVESTIGATION
OF CONNECTIVITY IN THE BRAIN
Kaminski M.
Department of Biomedical Physics, University of Warsaw, Warszawa,
Poland
In everyday practice of neurobiology research multivariate datasets
are analyzed. This concerns EEG, ECoG, MEG data but also can be
extended to fMRI and other recordings as well. Multichannel data require specific approach in order to fully explore information they contain. The issues and problems arising during analysis of such datasets
will be discussed. With special emphasis relations between signals
which describe influences between investigated structures will be
discussed. The concept of Granger causality will be introduced and
examples of estimators of causal influence of signals, especially the
Directed Transfer Function, and time-varying DTF (which describes
dynamical properties of transmissions between channels of the process) will be presented. The performance of the selected estimators
will be shown on simulated examples as well as on real EEG data.
POSTER SESSION I
TI: Development and Adult Neurogenesis
TI.01
SPATIAL MEMORY AND DIFFERENTIATION OF
ADULT HIPPOCAMPAL NEURONS: ROLE OF TIMING
Cestari V.1, Farioli-Vecchioli S.2, Saraulli D.1, Costanzi M.1,
Pacioni S.3, Cinà I.2, Aceti M.1, Micheli L.2, Bacci A.3, Tirone F.2
1
Institute of Neuroscience CNR, Piza, Italy; 2 Institue of
Neurobiology CNR, Rome, Italy; 3 European Brain Research Institute,
Rome, Italy
In the dentate gyrus adult neurogenesis plays a critical role in hippocampus-dependent spatial learning. However, how new neurons
become functionally integrated into spatial circuits and contribute
to learning and memory remains yet unknown. To study this issue, we used a mouse model in which the differentiation of adultgenerated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in
nestin-positive progenitor cells. This strategy selectively changes
the timing of differentiation of newly generated neurons without
affecting their number. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation, reduced dendritic arborization and spine
density. The genetic manipulation affected different hippocampusdependent learning and memory tasks and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional
analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows,
revealed that the new, adult-generated neurons up to 3–4 weeks of
age are required not only to acquire new spatial information but
also to use previously consolidated memories. Thus, the correct
unwinding of these key memory functions is critically dependent
on the correct timing of the initial stages of neuron maturation and
connection to existing circuits.
TI.02
INTERACTION OF SONIC HEDGEHOG SIGNALING
WITH PACAP IN CEREBELLAR GRANULE CELL
PROGENITORS
Niewiadomski P., Zhujiang A., Waschek J.
Neuropsychiatric Institute, UCLA, Los Angeles, CA, USA
Granule cells of the cerebellum arise from a secondary germinal zone, the external granule layer (EGL), where granule
cell progenitors (GCPs) proliferate in response to stimulation
with Sonic hedgehog (Shh). Deregulation of Shh signaling in
the cerebellum results in medulloblastoma (MB) in both mice
and humans. Pituitary adenylate cyclase-activating polypeptide
(PACAP), a ubiquitous and multifunctional polypeptide, is produced by the Purkinje cells of the cerebellum and by GCPs during development and binds to specific PAC1 receptors on GCPs.
It has been shown that (1) PACAP signaling prevents apoptosis
of GCPs exposed to mutiple insults, (2) it is capable of counteracting the proliferative effect of Shh on these cells and (3) its
deletion in ptc1+/- mice significantly increases MB incidence.
In the current study we present evidence suggesting that PACAP antagonizes Shh signaling in GCPs through activation of
292
protein kinase A. Our results also imply that PKA activation
is regulated by Shh- and PACAP-activated signaling pathways
independently of each other. We also show through microarray
analysis of cultured GCPs that PACAP and Shh activate distinct
sets of genes in these cells and that virtually all Shh-induced
changes in gene expression can be efficiently inhibited by 10
nM PACAP.
TI.03
THE EXPRESSION OF ERYTHROPOIETIN AND
ITS REGULATION DURING THE POSTNATAL
DEVELOPMENT OF THE RAT RETINA
Szabo A.1, Végvári D.1, Magyar A.1, Deák G.2, Lukáts Á.1,
Berta Á.1, Szél Á.1
1
Department of Human Morphology and Developmental Biology,
2
Eye Clinic, Semmelweis University, Budapest, Hungary
In the present study, we examined developing postnatal and adult
rat retinas by immunohistochemistry and western blot in order to
identify the temporal and spatial onset of the expression of EPO
and its presumptive regulating factors (HIF1-α and HIF2-α). In the
adult the EPO was expressed in the photoreceptor inner segments,
in the inner nuclear, ganglion (GCL), and both plexiform layers. At
birth, we found strong immunoreactivities in the GCL and in the
inner part of the neuroblast layer and weak staining in the outer
part of the neuroblast layer. During the following days the immunoreactivity of the ganglion cells and presumptive amacrine cells
became more prominent. From the middle of the first week strongly
stained horizontal cells were observed at the level of the separating OPL. The WB analysis resulted high protein levels in the developing retina samples and low protein amounts in the adult. Both
hypoxia-induced factors showed similar expression pattern, being
expressed in the inner retinal layers. At birth, only the HIF1-α was
detectable. The HIF2-α was not detectable until the second half of
the second postnatal week and its expression remained weak. The
early appearance and distribution of the erythropoietin correlates
in time with the main apoptotic processes of the postanatal rat
retina. Since the EPO exerts neuroprotective properties in neural
tissue, we hypothesize that it acts as a regulating factor during the
retinal development.
TI.04
EFFECT OF OLFACTORY STIMULATION ON
NEUROGENESIS IN THE ROSTRAL MIGRATORY
STREAM
Martoncikova M., Lievajova K., Orendacova J., Racekova E.
Institute of Neurobiology SAS, Kosice, Slovak Republic
The subventricular zone retains the capacity to generate new
neurons from stem cells. The newly generated cells migrate
along the rostral migratory stream (RMS) to the olfactory bulb
(OB) where they differentiate into interneurons. The OB is the
fi rst relay on the olfactory sensory pathway. We have previously shown that the number of proliferating cells in the RMS of
young rats depends on their age. The aim of this study was to
investigate if olfactory stimulation (OS) can influence the number of proliferating cells in the RMS of young rats. Since it is
known that nitric oxide have antiproliferative action we have
also studied occurrence of nitrergic NADPH-d positive cells in
the vicinity of proliferating cells. We used 15 natural or synthetic odorants to stimulate olfaction. The rats were olfactory
stimulated from the day of their birth (postnatal day 0 – P0) for 2
weeks (P14 group) or for 3 weeks (P21 and P28 groups). Control
rats were alike divided into three groups according to the age.
To label proliferating cells in the RMS, the rats were injected
with proliferation marker – bromdeoxyuridine (BrdU). Brain
sections were processed for BrdU immunohistochemistry and
NADPH-d histochemistry. We found that OS significantly increased the number of proliferating cells in the RMS of young
rats and concurrently number of nitrergic cells decreased. These
results indicate positive effect of OS on postnatal neurogenesis
in the RMS. Support: VEGA grants 2/0147/09; 2/0058/08.
TI.05
NEUROGENESIS AND OLFACTORY-GUIDED
BEHAVIOR IN ADULT LABORATORY OPOSSUMS
(MONODELPHIS DOMESTICA)
Grabiec M., Djavadian R., Turlejski K.
Department of Molecular and Cellular Neurobiology, Nencki Institue
Activation of the receptor 5-HT1A increases the rate of neurogenesis in the subventricular zone (SVZ) and its blocking reduces
it. Cells generated in the SVZ settle in the olfactory bulb as its
interneurons. We investigated in laboratory opossums if changes
in the rate of SVZ neurogenesis influence olfactory detection of
food and discrimination of conspecific odors. Four groups of one
year old opossums were tested. The fi rst group was injected i.p.
for 7 days with saline, the second group with 5-HT1A receptor agonist 8-OH-DPAT, the third group with partial agonist buspirone
and the fourth group with the antagonist, WAY100635. BrdU was
injected as a marker of newly generated cells. One month later
opossums had to locate crickets hidden in the litter. Pretreatment
with WAY100635 significantly extended time needed for location
of the hidden food in comparison to other groups. Next opossums
were exposed for two days to samples of urine of two other opossums. Time of investigation of samples decreased with experience. Opossums injected with buspirone or WAY100635 investigated probes for a longer time and buspirone-treated approached
them more frequently. There were no preferences for investigation of new odors. Post mortem investigation of the numbers of
BrdU-labeled cells showed predictable differences. These results
indicate that reduction of the rate of neurogenesis hampers olfactory detection of food, but detection of odors of conspecifics is
influenced in more complex way.
TI.06
NEURAL XENOTRANSPLANTATION: THE FATE OF
HUMAN UMBILICAL CORD BLOOD NEURAL STEM
CELLS (HUCB-NSC) AND HOST RESPONSE TO BRAIN
INJURY AND CELL GRAFT
Jablonska A., Kozlowska H., Wanacka E., Winiarska H.,
Domanska-Janik K., Lukomska B.
NeuroRepair Department, Mossakowski Medical Research Centre
PAS, Warszawa, Poland
Neural stem cells (NSC) are potentially a promising treatment
strategy for neurological diseases. There is a proof that intrac-
erebral grafting of NSC can have restorative effects. Nevertheless, a major obstacle is the induction of a host-derived immune
response followed by graft injection. The goal of the study was
to analyze the fate of HUCB-NSC transplanted in injured rat
brain. Methods: 2×10 4 HUCB-NSC were tx into corpus callosum
of a focal brain injury induced by OUA injection (1 μl/50 nmol)
into striatum of adult Wistar rats. After 1, 3, 7 and 14 days brains
were removed and analyzed immunocytochemically. Results.
One day after HUCB tx, most cells remained in the injection
site with few cells migrated to the lesion area. Concomitantly,
infiltration of ED1+ and CD15+ cells with occasional appearance
of CD5+ and CD45+ cells was seen. At 3rd day some HUCB-NSC
in graft core expressed neuronal (NF-200) or astrocytic (GFAP)
markers. Migration of tx cells into the lesion was observed. At
7th day HUCB-NSC were found close to injured area. By 14 days,
no viable HUCB-NSC have been noticed with cellular debris in
graft core. Limited number of ED1+, CD15+, CD5+ and CD45+
cells were found most likely due to HUCB-NSC rejection. Conclusions. Massive loss of transplanted HUCB-NSC was probably
due to post traumatic inflammation and acute immune reaction
of the host in term of cross-species grafting paradigm. Supported by MSHE grant N401 014235 and Fondation Jerome Lejeune
scientific grant.
TI.07
SPINAL CORD INJURY OR ENHANCED PHYSICAL
ACTIVITY STIMULATE DIVISION OF EPENDYMAL
CELLS IN RAT SPINAL CORD
Novotna I.1, Slovinska L.1, Cizek M.2, Nagyova M.1, Rosocha J. 3,
Radonak J.4, Vanicky I.1, Cizkova D.1
1
Institute of Neurobiology SAS, Kosice, Slovakia; 2 Department
of Infectious Diseases, University of Veterinary Medicine, Kosice,
Slovakia; 3 Associated Tissue Culture Bank, Medical Faculty,
4
II Surgical Clinic, University of PJ Safarik, Kosice, Slovakia
Ependymal cells (EC) in the spinal cord central canal (CC) are
believed to be responsible for the postnatal neurogenesis following pathological or stimulatory conditions. In the present
study we have analyzed the proliferation of the CC EC in adult
rats processed to spinal cord injury (SCI) or enhanced physical
activity. To label dividing cells, a daily injection of Bromodeoxyuridine (BrdU) was administered over a 14 days. Quantification of BrdU positive EC was performed by using stereological principles of systematic random sampling and optical
Dissector software. The number of BrdU labeled EC increased
gradually with the time of survival after both paradigms, SCI
or increased physical activity. In the SCI group we have found
8.2-fold (7 days) and 11.3-fold (14 days) increase of proliferating EC in the rostro-caudal regions. Furthermore, the cervical
spinal cord segments revealed 2 to 3-fold increase of EC for
both time-points analyzed. In the second group subjected to
enhanced physical activity by running wheel, we have observed
1.8 fold increase of dividing EC in the lumbar and 3.2 fold increase in the cervical spinal cord segments at 7 days, but no
significant progression at 14 days. This data shows, that SCI or
enhanced physical activity in adult rats induces an endogenous
EC response leading to their increased proliferation, which
may be beneficial for recovery of motor function. Supported by:
APVV 51-002105, VEGA 2-0019-08, VEGA 1-0674-09, VEGA
1/4223/07, APVV SK-CZ-0045-07.
TI.08
GUIDING CELL-BASED THERAPY FOR
NEUROLOGICAL DISEASES WITH NONINVASIVE
CELLULAR IMAGING
Walczak P.1, Gorelik M.2, Levy M.2, Rumpal N.2, Rifkin R.2,
Muja N.1, Kim H.1, Kerr D.2, Bulte J.1
1
Department of Radiology, 2 Department of Neurology, Johns
Hopkins University, Baltimore, MD, USA
Taking advantage of recent developments in molecular biology
and imaging, we have developed a system for monitoring status of
transplanted cells non-invasively. We use high-resolution MRI to
acquire information about the position of grafted cells and reporter
gene-based bioluminescent imaging (BLI) to monitor their survival and differentiation. Here, we report on two different approaches
for the in vivo imaging of glial precursors cells (GRPs). Approach
I: Monitoring of the targeted, intravascular cerebral delivery of
GRPs. Cells were engineered to express VLA-4 integrin (to enhance vascular adhesion) and were labeled with the MR contrast
agent Feridex. Recipient rats were injected i.p. with lipopolysaccharide, a known inducer of endothelial VCAM-1 expression, and
the cells were infused into the carotid artery. MRI demonstrated
extensive hypointense regions, indicating successful targeting.
Approach II: Monitoring of the survival and differentiation of intracerebrally injected GRPs. Cells were engineered to express luciferase under the control of a constitutive or the cell-type-specific
promoters and were injected into the brain of immunodeficient or
immunocompetent mice. BLI demonstrated that transplanted GRPs
survived for extended periods of time in immunodeficient animals,
while, in immunocompetent animals, rejection was initiated two
weeks after grafting. With cell type-specific promoters, we were
able to visualize the process of glial differentiation in vivo.
TI.09
RELATIONSHIP BETWEEN EXTRACELLULAR
MATRIX COMPONENTS AND MMPS ACTIVITY
DURING DEVELOPMENT OF NEURAL STEM CELLS
FROM UMBILICAL CORD BLOOD (HUCB-NSC)
Szymczak P., Sypecka J., Zalewska T.
Accumulating evidence indicates that extracellular matrix components and metalloproteinases play a pivotal role in the development
of stem cells. The aim of our study was to check if the presence of
laminin, fibronectin, and collagen affect MMPs activity (MMP-9 and
MMP-2) which in turn might enhance cell proliferation and differentiation. The cells of HUCB-NSC were cultured without serum for two
weeks on extracellular matrix components-coated plates. On 4th, 8th,
and 14th day we performed proliferation assay, and determination of
MMPs activity (in situ zymography) followed by immunocytochemistry with specific neural markers. Our results show that among all
of the investigated ECM components fibronectin stimulated most intensively cell proliferation and differentiation, especially toward neurons. We also observed the increase of MMPs activity (~20% increase
in the 2-weeks culture) in the presence of fibronectin. To confirm the
influence of MMPs on developmental processes of neural stem cells
we used inhibitors of MMPs – GM6001, and doxycycline. We found
that the inhibition of MMPs by GM6001 decreased cell proliferation
(~30%) and differentiation into neurons (~20%). As it was shown in
294
the present study the fibronectin occured to be the potent factor in promoting cell proliferation and differentiation and support the idea that
MMPs may contribute to the mechanism involved in the development
of neural stem cells. Supported by MSRHE grants: 1266/P01/2006/31
and N40101832 /0296.
TI.10
HUCB-NSCS GROWTH CONTROL BY
BIOFUNCTIONALIZED SURFACES
Zychowicz M.1, Ruiz A.2, Ceriotti L.2, Lisboa P.2, Mehn D.2,
Coecke S.3, Rausher H.2, Colpo P.2, Rossi F.2, Domanska-Janik K.1,
Buzanska L.1
1
NeuroRepair Department, Mossakowski Medical Research
Centre PAS, Warszawa, Poland; 2 Nanobiosciences Unit, 3 In Vitro
Toxicology Methods Unit, Institute for Health and Consumer
Protection, European Commission, Joint Research Centre, Ispra, Italy
Cell growth platforms with biofunctionalized surfaces were fabricated to control and direct HUCB-NSC fate decisions. Two different nano/micro techniques: microcontact printing and piezoelectric
non-contact spotting were used to allocate biomolecules (poly-Llysine and fibronectin) on cell-repellent, non adhesive substrate. Such
methods allow controlling the spatial distribution and content of the
biomolecules on the microarray and governing cell adhesion in unspecific (electrostatic) or specific (receptor-mediated) manner. Patterning of biomolecules in different conditions on the single growth
platforms enables to influence and compare stem cells developmental
processes (proliferation /differentiation) at variable environments. To
reflect/mimic stem cell niche we applied functional domains containing ECM protein spotted together with the small signaling molecules
(notch, wnt, shh). Such approach enable directing of neural stem cell
developmental program by inducing intracellular molecular pathways leading to either self renewing- or differentiating- (neuronal or
astrocytic) state. We characterized active biofunctionalized domains
on fabricated microarrays by applying two methods of surface analysis: ellipsometry measurement and surface plasmon resonance system. This type of bioengineered cell growth platforms can be used
for screening the mechanisms governing neural stem cell fate decisions and adverse reactions upon environmental stimuli. Grant No
0141/B/ P01/2008/35.
TI.11
THE PHENOTYPE OF NEWLY GENERATED CELLS
IN THE ADULTS’ BRAIN OF SOME SPECIES OF
RHINOLOPHIDAE
Ghazaryan A.1, Djavadian R.2, Yavrouyan E.1, Turlejski K.2
1
Department of Biology, Yerevan State University, Yerevan,
Armenia; 2 Molecular and Cellular Neurobiology, Nencki Institute
In the brain of adult mammals neurogenesis continues in two regions:
the dentate gyrus (DG) and subventricular zone of the lateral ventricles (SVZ). Here we present data concerning neurogenesis in brains
of Rhinolophus (Rh) ferrumequinum, Rh euryale, Rh mehely and Rh
hipposideros. These bats were captured on permission of the Ministry of Environment of Armenia. After capture all bats were injected
with bromodeoxyuridine (BrdU, 300 mg/kg). Seven to nine days later
animals were perfused transcardially with 4% paraformaldehyde in
narcosis. We used immunohistochemical double-labeling to char-
acterize the phenotype of newly generated cells. Colocalization of
BrdU with NeuN (marker of mature neurons), glial fibrillary acidic
protein (GFAP, astrocytic marker) and 2`,3`cyclic nucleotide phosphatase (CNP, oligodendrocytic marker) was examined using confocal microscope. We found that in both neurogenic regions the rate of
neurogenesis was highest in Rhinolophus ferrumequinum and lowest
in Rh mehely. Double-immunolabeling showed that in all neurogenic
regions of the bats’ brain the neuronal phenotype dominated among
newly generated cells, while proportion of astrocytes was low. BrdU
colocalized with CNP in only a few cells in the SVZ of Rh ferrumequinum. We suggest that the rate of neurogenesis in different species may
depend on the bats’ ecology.
TI.12
TRANSPLANTATION OF NNC1 CELLS AFTER
EXPERIMENTAL STROKE
Braun H.1, Baldauf K.1, Paul G.2, Brundin P.2, Reymann K.1
1
Department of Neuropharmacology, Leibniz Institute for
Neurobiologiy, Magdeburg, Germany; 2 Department of Neuronal
Survival Unit, Wallenberg Neuroscience Center, Lund, Sweden
Transplantation of stem cells is currently investigated as an option
for treatment of stroke. In this study we transplanted adult human
cells NNC1 derived from brain biopsy after experimental stroke in
rats. Transient ischemia in rats was performed by the filament model
of middle cerebral artery occlusion (fMCAO). About 3×105 NNC1
cells were transplanted near to the necrotic area 7 days after fMCAO and the brain of treated rats was investigated 4 weeks after
transplantation. Rats were immunosuppressed by daily injections of
cyclosporine A, prednisolone and azathioprine. Grafted cells were
localized by anti-human specific antibodies HuNu and MTC02.
Cells appeared to be located dispersed, however there was almost no
cell-migration. The number of localized NNC1 cells 4 weeks after
transplantation was rather limited indicating a reduced survival rate
which was probably caused by host versus graft rejection processes.
The latter is reflected by activation of Ox42 and ED1 positive microglia/macrophages. Also, we detected CD8 and TCR positive T-cells
infiltrating to the necrotic area. Immunocytochemical analysis revealed expression of nestin and βIII-tubulin by grafted NNC1. Interestingly, some βIII tubulin expressing NNC1 were found to be located in blood vessels. In conclusion, both the survival and the capacity
to neuronal differentiation of grafted NNC1 cells are limited.
TI.13
ROBUST IN VITRO MIGRATION OF HUMAN
UMBILICAL CORD BLOOD NEURAL STEM CELLS
(HUCB-NSC) TOWARD INFARCTED BRAIN TISSUE
Janowski M., Lukomska B., Domanska-Janik K.
Potentially therapeutic neural stem cell line from human cord blood
(HUCB-NSC) has been established in our laboratory. Reaching appropriate target by transplanted cells is a prerequisite for success of
cell therapy for stroke. The question arises what is the migration potential of HUCB-NSC towards infarcted brain tissue. The migration
of HUCB-NSC towards rat tissue homogenates from healthy brain
(THHB) and ouabain-induced focal brain injury (THIB) obtained 6
h, 48 h and 7 days after insult was studied in vitro using transwells.
Additionally the migratory activities of HUCB-NSC was checked in
the presence of migration inducing proteins IGF-1 (200 ng/ml) and
SDF-1 (10 ng/ml) dissolved in culture medium. Immunocytochemical analysis of migration-related receptors (CXCR-4, IGF-1R) on
HUCB-NSC was performed. Results: Immunohistochemistry of
HUCB-NSC unveiled expression of CXCR-4, IGF-1R. HUCB-NSC
revealed robust migration toward THIB in comparison to THHB,
which was most pronounced in the presence of 48 h postinfarct
brain tissue (900 vs. 300 cells/well, P<0.05). The presence of IGF-1
and SDF-1 in medium increased significantly HUCB-NSC migration but the effect was much weaker in comparison to injured brain
tissue. The ability of robust in vitro migration of HUCB-NSC towards infarcted rat brain tissue has been confirmed. Neither IGF-1
nor SDF-1 seems to play a pivotal role in this lesion-induced migration of HUCB-NSC. Supported by MSHE grant: N N401 332636.
TI.14
CLIP-170 AND IQGAP1 IN DENDRITIC ARBOR
DEVELOPMENT
Swiech L., Blazejczyk M., Jaworski J.
Laboratory of Molecular and Cellular Neurobiology, International
Institute of Molecular and Cell Biology, Warszawa, Poland
The precise control of the microtubule polymerization dynamics
as well as strict actin organization are both crucial for formation of
neuronal dendritic arbor and require microtubule and actin binding
proteins activity. Cytoplasmic linker protein 170 (CLIP-170), one
of microtubule plus-end binding proteins, regulates microtubule
dynamics at plus-end during polymerization, by promoting rescuephase in its phosphorylation status dependent manner. We show
evidence that mammalian target of rapamycin (mTOR), is one of
kinases capable of regulating CLIP-170 activity and both, mTOR
and CLIP-170 are crucial for proper dendritic arbor development
of hippocampal neurons. Furthermore, we identified in neurons
several proteins, which bound to CLIP-170 when mTOR is active,
including IQGAP1, a known partner of CLIP-170 and regulator
of the actin dynamics. Taken together our data strongly suggest
that CLIP-170 activity during dendritogenesis can be regulated by
mTOR at the level of CLIP-170 protein-protein interactions. Moreover, obtained results, showing mTOR dependent interaction of
CLIP-170 with IQGAP imply that mTOR can coordinate tubulin
and actin cytoskeleton organization. Supported by Polish Ministry
of Science and Higher Education Research Grant 2P04A01530 and
Polish-Norwegian Research Found grant PNRF-96-AI-1/07.
TI.15
DYNAMIC RELATIONSHIPS OF CONVENTIONAL
AND NEURON-SPECIFIC ENDOSOMAL REGULATORS
IN DENDRITES
Lasiecka Z., Yap C., Winckler B.
Department of Neuroscience, University of Virginia, Charlottesville,
VA, USA
The unique functions and morphology of neurons require a specialized endosomal system that recycles, sorts, and targets proteins.
The neuronal endosome is unique in that it is polarized into somatodendritic and axonal domains, has neuronal-specific regulators,
and is modulated by generic regulators that confer neuronal-specific
properties. NEEP21, a neuronal-specific early endosomal regulator,
and EEA1, a generic early endosomal regulator, are polarized to somatodendritic domain and bind syntaxin13 (stx13), which occupies
early/recycling endosomes in somatodendritic and axonal domains.
NEEP21 and stx13 regulate AMPA receptor recycling; NEEP21 regulates trafficking of L1 cell adhesion molecule. It is not known how
these endosomal regulators function together to transport cargo in
neurons. Due to the dynamic nature of the endosomal system, live
imaging is required to understand the relationships and dynamics of
different compartments. We carried out live imaging of GFP/RFPtagged regulators in cultured hippocampal neurons. NEEP21 occupies stationary (containing L1) and dynamic (devoid of L1) round
compartments. EEA1-containing round and static compartments do
not significantly colocalize with NEEP21, but often are adjacent to it.
Stx13 resides in dynamic, elongated carriers and in round stationary
compartments where it partially colocalizes with EEA1 and NEEP21.
We interfered with function of stx13 using expression of dominantnegative construct and found that it affects L1 trafficking.
TI.16
RNA-BINDING PROTEINS IN DENDRITOGENESIS
Perycz M., Parobczak K., Jaworski J.
Proper development of dendritic arbor is a precisely orchestrated
process in which translation is shown to play pivotal role. RNA
binding proteins such as ZBP1 (beta-actin zipcode binding protein 1), Staufen1 and Staufen2 bind to and transport their target
mRNAs, enabling their local translation in dendrites and taking
part in regulation of this process. ZBP1 knockdown with short interfering RNAs in developing rat hippocampal neurons in vitro,
resulted in decrease in total number of dendrites and dendritic
tree complexity, which was reversed both with siRNA-resistant
ZBP1 rescue mutant and jasplakinolide treatment that prevents
depolymerisation of actin cytoskeleton. In contrast, mature neurons with stable dendritic arbors, did not lose dendrites upon ZBP1
depletion, which suggests ZBP1 plays role in formation of new
dendrites as opposed to maintenance of mature ones. We also
show that both Staufen1 and Staufen2 knockdown in developing
neurons results in impoverishment in dendritic arbors complexity
and decrease in total number of dendrites. RNA-binding proteins
can be subject to phosphorylation, which modulate their activity,
and one possibility for a common regulator of their role in dendritogenesis would be Src kinase, which we show to phosphorylate
ZBP1 and Staufen1. Taken together, presented data demonstrate
that mRNA transport and possibly local protein synthesis play a
prominent role in development of dendritic arbor. Ministry of Science and Higher Education Grant NN301314733
TI.17
FUNCTIONS OF TGF-β2 AND GDNF IN THE
DEVELOPMENT OF THE MOUSE NERVOUS SYSTEM:
EVIDENCE FROM DOUBLE MUTANT MICE
Rahhal B.1, Heerman S.2, Krieglstein K.2
1
Faculty of Pharmacy, An-Najah National University, Nablus,
Palestine; 2 Faculty of Medicine, University of Freiburg, Freiburg,
Germany
A major area of investigations in neuroscience is directed at
understanding factors that participate in neuronal survival and
296
death. Therefore they are good candidates to be responsible
for different neurodegenerative diseases such as Alzheimer’s,
and Parkinson’s diseases. Many recent advancements in our
laboratory have revealed that growth factors acting in synergy
can regulate neuronal survival much more effectively than individual factors alone. Impairment of neuronal survival is the
cause of a considerable number of neurodegenerative diseases.
TGF-β has been shown to act in synergy with neurotrophic
factors, most strikingly in combination with GDNF. Since no
information was available about the overall significance of
TGF-β/GDNF cooperation and/or synergism for nervous system development, the present study was undertaken to test the
paradigm of the developmental requirement of growth factor
synergism and cooperation. Double knockout mice that lack
both TGF-β2 and GDNF were generated. Neuron populations
analyzed included all those in which biological effects of either growth factor had been documented before. Finally, an
understanding of the role of TGF-β/GDNF synergism in vivo
through the analysis of the nervous system development of
the corresponding double mutant mice can provide significant
basic and clinical insight into mechanisms that may lead to
overcoming neuron loss and verify potential targets for neurological diseases.
TI.18
A QUANTITATIVE STUDY OF THE GLIAL
CELL RESPONSE TO BRAIN INJURY AFTER
INTRACEREBRAL AND INTRAVENOUS
TRANSPLANTATION OF BONE MARROW STROMAL
CELLS
Opydo-Chanek M.1, Pasiut S.2
1
Department of Experimental Hematology, Institute of Zoology,
Jagiellonian University, Kraków, Poland; 2 Department of Clinical
Rehabilitation, Academy of Physical Education, Kraków, Poland
Experimental studies have shown that bone marrow stromal
cells (BMSCs) promote functional recovery after traumatic
brain injury. The beneficial effects of BMSCs therapy were
demonstrated after intravenous, intraarterial and intracerebral
transplantation of these cells. A reasonable explanation for
the benefit provided by BMSCs is that these cells stimulate
regenerative changes in the damaged brain, including glial
remodeling. The aim of the present study was to determine
and compare the time course of the astrocyte and microglial/
macrophage cell reaction to cerebral cortex injury after intracerebral and intravenous administration of BMSCs. Female
Wistar rats were subjected to cerebral cortex injury followed
by the injection of BMSCs or PBS directly to the injury site or
to the tail vein. Using histology and immunohistochemistry,
the distribution of astrocytes and microglia/macrophages was
analyzed in the injured cerebral cortex. BMSCs treatment affected glial cell response to brain injury. The effects of BMSCs
activity were dependent on the site of their administration. Intracerebral transplantation of BMSCs showed significant effect on the astrocyte response whereas intravenous route of
BMSCs administration increased especially the microglia/
macrophages number in the injured brain. The results suggest
that implanted cells can enhance the restorative processes in
the injured brain through the stimulation and modulation of the
cellular response to injury.
TI.19
FUNCTIONAL AND MORPHOLOGICAL ANALYSIS
OF DRG NEURONS UNDER L-ARGININE APPLICATION
AFTER PNS INJURY
Guseva D., Nigmetzyanova M., Masgutova G., Chelyshev Y.
Department of Histology, Kazan State Medical University, Kazan,
Russia
Over the past 30 years, the role of nitric oxide (NO) in biology
has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell
communication and signal transduction. NO can play a double
role after injury of the PNS and CNS in accordance with its
concentration, time of synthesis and type of NO-synthase participating in its production. Three isoforms of NOS have been
reported – nNOS, eNOS and iNOS. They are reaching greatest
expressions during the 2nd week, and the dynamic expression of
individual isoforms is different. Since the protective or toxic
effects of NO depend on its concentration, the cell type it is
expressed, it is useful to apply donors or inhibitors of NOS in
restrict time points after injury. It was established previously
that increasing expression of iNOS results in death of neurons but nNOS plays neuroprotective role after PNS lesion. It
is plausible that producing of NO with participation of nNOS
provide the amount of NO which serves as a neuroprotective
signal for neurons after injury. In this study, we studied the
expression of caspase-3 in different population of DRG neurons
using sciatic nerve transection with the purpose to explore the
potential functional link between locally applied donor of NO
and the ability of sensory neurons to survive. We observed better functional recovery in animals treated with L-arginine 7
days after nerve injury and decreasing of caspase-3 expression
in DRGs L4-L5.
TII: Neural Excitability, Synapses and Glia:
Cellular Mechanisms
TII.01
ANTIDEPRESSANT-LIKE FEATURES OF MICE
WITH TRANSGENIC ACTIVATION OF Ras IN BRAIN
NEURONS
Leske O.1, Bichler Z.2, Heumann R.1
1
Department of Molecular Neurobiochemistry, Ruhr University
Bochum, Germany; 2 Prous Insititute for Biomedical Research,
Barcelona, Spain
Brain-derived neurotrophic factor (BDNF) is implicated in
clinical depression. Thus, BDNF expression is enhanced by antidepressants while stress exposure and depression decreases
it. A major BDNF intracellular signalling pathway is the Rasextracellular signal-regulated kinase (ERK) cascade. Here,
we test its possible contribution on antidepressant activity by
utilizing a synRas transgenic mouse model expressing constitutively activated human Ha-Ras in differentiated neurons
[Heumann et al. (2000) J Cell Biol 151: 1537]. Immunoblotting analysis in hippocampus and cortex revealed that chronic
fluoxetine administration to C57Bl/6 mice led to an increased
activation of endogenous Ras and activating ERK1/2 phosphorylation. SynRas mice exhibited chronically increased levels
of activated Ras and activating ERK1/2 phosphorylation in the
cortex and hippocampus. This was clearly associated with an
antidepressant-like behaviour in four different animal models
of depression. Furthermore, restraint stress-induced corticosterone response was attenuated in synRas mice, throughout
stress and recovery time period. Because adult neurogenesis
may play a role in depression we assessed the proliferation of
hippocampal progenitors. Here we show that proliferation was
not correlated with the antidepressant activity in synRas mice.
Taken together, an antidepressant state was established in a genetic model of enhanced neuronal Ras signalling without correlation to hippocampal precursor cell proliferation.
TII.02
HIPPOCALCIN SIGNALING IN SPINES
OF HIPPOCAMPAL NEURONS
Cherkas V.1, Dovgan A.1, Fitzgerald D.2, Tepikin A.2,
Burgoyne R.2, Belan P.1
1
Department of General Physiology of the Nervous System,
Bogomoletz Institute of Physiology, Kiev, Ukraine; 2 Department of
Physiology, University of Liverpool, Liverpool, UK
Hippocalcin (HPCA) is a Ca 2+-binding protein, and its Ca 2+-dependent activation in hippocampal neurons is one of the necessary steps involved in many signal transduction mechanisms.
In this work we have examined if synaptic glutamate receptor
activation can result in HPCA signaling in spines of hippocampal neurons. Spontaneous and evoked synaptic activity induced
excitatory postsynaptic potentials (EPSPs) in the hippocampal
neurons leading to AP bursts and HPCA-YFP translocation
to many sites in a neuronal dendritic tree including dendritic
spines. In neurons clamped at −40 mV, episodes of presynaptic
activity resulted in EPSCs associated with HPCA translocation
mainly to dendritic spines while no translocation was observed
at −70 mV . These results indicate that synaptic NMDAR activation is necessary for HPCA-YFP translocation. T- and R- rather
than L-types of voltage activated Ca 2+ channels also contribute
to the observed translocation. FRET measurements between
HPCA tagged by Yellow and Cyan Fluorescent Proteins have
shown that the translocation was due to HPCA-FPs insertion in
patches of spine membrane resulting in decrease of protein concentration in the cytosol of spines and diffusion of new HPCAFP molecules from the dendritic trunk. Thus, we have shown
that hippocalcin may signal as a coincident detector in spines of
hippocampal neurons by means of its robust insertion in spine
plasma membrane.
TII.03
EFFECTS OF CHRONIC FLUOXETINE TREATMENT
ON CATALEPSY, IMMUNE RESPONSE AND BRAIN
SEROTONIN SYSTEM IN MICE GENETICALLY
PREDISPOSED TO CATALEPSY
Tikhonova M.1, Idova G.2, Kulikov A.1, Popova N.1
1
Laboratory of Behavioral Neurogenomics, Institute of Cytology
and Genetics SB RAS, Novosibirsk, Russia; 2 Laboratory of the
Mechanisms of Neurochemical Modulation, State Research Institute
of Physiology SB RAMS, Novosibirsk, Russia
ASC/Icg (Antidepressant Sensitive Catalepsy) mouse strain
selected for high predisposition to pinch-induced catalepsy is
characterized by depressive-like behavior and impaired immune response. Chronic treatment with SSRI fluoxetine attenuated catalepsy manifestation and normalized a decreased
number of rosette-forming cells (RFC) in spleen in ASC mice.
Chronic fluoxetine administration had no effect on catalepsy
and RFC number in mice of parental cataleptic CBA/Lac strain.
Fluoxetine failed to alter 5-HT1A receptor functional activity in
mice of both strains and diminished 5-HT2A receptor functional
activity in CBA but not in ASC mice. No effect on cortical
5-HT1A and 5-HT2A receptor mRNA levels and on 5-HT1A receptor, tph2 (tryptophan hydroxylase-2) and SERT (serotonin
transporter) mesencephalic gene expression was observed in
ASC mice. The data evidenced the validity of ASC mouse
strain as prospective model for research of psychoneuroimmune mechanisms of antidepressant effects and development
of depressive-like state.
TII.04
LONG-TERM POTENTIATION IN HIPPOCAMPAL
BASAL DENDRITES: ROLE OF DIFFERENT
MODULATORY SYSTEMS
Ramachandran B., Parvez S., Frey J.
Department of Neurophysiology, Leibniz Institute for Neurobiology,
Magdeburg, Germany
Long-term potentiation (LTP) and long-term depression (LTD) are
considered to be cellular models suitable for studying the synaptic
changes that likely occur during learning and memory. LTP has
distinct phases, a transient protein synthesis-independent stage
(early-LTP) followed under distinct circumstances by a late, longlasting and protein synthesis-dependent stage (late-LTP). In hippocampal CA1 neurons, LTP in either the apical or basal dendrites
differ in their molecular requirement during induction as well as
298
the setting of the tag, for example CaM kinase II mediates the setting of the tag in stratum radiatum but in basal dendrites (stratum
oriens) the setting of the tag is mediated by either protein kinase
A or protein kinase M zeta (Sajikumar et al. 2007). It has been reported that the late-LTP in the CA1 stratum radiatum requires the
synergistic activation of different neurotransmitters during its induction (Frey 1997, Frey and Morris 1998). From this background
we now investigated whether the LTP in stratum oriens requires
similar or other synergistic interactions of different modulatory
systems when compared with the stratum radiatum. Our preliminary studies using different selective antagonists of distinct modulatory transmitters systems revealed that late-LTP in basal dendrites is different with respect to its requirements for its induction
when compared with late-LTP in apical CA1-dendrites.
TII.05
NEURON TYPE RECONFIGURATION OF GABA-ERGIC
TONIC INHIBITION IN MOUSE BARREL CORTEX
INDUCED BY SENSORY LEARNING
Urban-Ciecko J., Mozrzymas J.
Department of Biophysics, Wroclaw Medical University, Wrocław,
Poland
Pairing tactile stimulation of a row of whiskers with a tail shock
results in expansion of the cortical representation of stimulated
vibrissae accompanied by increase of the GABAergic markers.
Using the same paradigm, we examined exstrasynaptic GABA A
receptors-mediated tonic inhibition. Whole-cell patch-clamp recordings were performed in layer IV neurons and tonic current
was assessed by application of the GABA A receptors blocker-picrotoxin (PTX) or by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin3-ol (THIP, a superagonist at delta-subunit containing GABA A
receptors) in ex vivo slices of the barrel cortex in adult mice. PTX
and THIP produced greater shifts in baseline holding current in
excitatory cells within the cortical representation of “trained”
vibrissae, visible 24 hours after the end of training. Recordings
from layer IV fast spiking neurons showed that the training induced smaller baseline shifts after the application of both substances. Regular spiking non-piramidal neurons had similar shifts
in baseline holding current in both control and trained mice. These
data indicate that associative learning paradigm results in a neuron type reconfiguration in tonic inhibition of layer IV neurons
situated within the barrel representing stimulated vibrissae. This
reconfiguration can be caused by changes in the level of deltasubunit containing GABA A receptors. Supported by the Ministry
of Science and Higher Education grants PBZ/MNiSW/07/2006/02
and N401 028 32/0664.
TII.06
GLUTAMATE TRANSPORTERS IN RAT BRAIN
EXPOSED TO POLYCHLORINATED BIPHENYLS
AND POLIBROMINATED FLAME RETARDANTS
Sulkowski G.1, Struzynska L.1, Dabrowska-Bouta B.1,
Albrecht J.2, Lazarewicz J.1
1
Department of Neurochemistry, 2 Department of Neurotoxicology,
Mossakowski Medical Research Centre PAS, Warszawa, Poland
Polychlorinated biphenyl (PCB) congeners and polibrominated
flame retardants (BFRs) are environmentally occurring toxins that
exhibit a broad range of adverse biological effects including neurotoxicity. Due to their stability and lipophilic character, they persist
in the environment and accumulate in brain of animals and humans contributing to induction of neurological disorders. The
mechanisms by which PCBs and BFRs cause neurotoxic effects
are still not completely understood. However, it is postulated that
glutamate excitotoxicity may be involved. The main glutamate
transporters GLT-1 and GLAST are membrane-bound proteins
localized in glial cells. The clearance of synaptically released
glutamate by these proteins protects neurons from excitotoxicity.
The aim of the study was to establish whether the chronic exposure to these substances may influence the expression and activity of main glutamate transporters in rat brain. Aroclor 1254
(PCBs) and tetrabromobisphenol A (BFRs) were administered by
oral gavage, which resembles the human exposure through the
food chain, for two weeks. Significant changes in the expression
of glutamate transporters were observed, especially in the case
of GLT-1. The effect was more pronounced for Aroclor 1254.
The results suggest that GLT-1 is a molecular target of this toxin
that may, almost partially, contribute to PCBs-induced excitotoxicity. This study was supported by grant nr NN401024635 from
Polish Ministry of Science and Higher Education.
TII.07
BRAIN LEVEL OF FKBP-51, A GLUCOCORTICOID
RECEPTOR COCHAPERONE, IS DECREASED
IN A NEURODEVELOPMENTAL ANIMAL MODEL
OF SCHIZOPHRENIA
Basta-Kaim A., Budziszewska B., Kubera M., Leskiewicz M.,
Regulska M., Korzeniak B.
Recent studies suggest that dysregulation of the HPA axis activity and disturbance in glucocorticoid receptor (GR) action are
involved in the pathogenesis of schizophrenia. The hyperfunction of GR can result from alterations in GR phosphorylation
status or numbers of its cochaperones. The most important GR
cochaperone FKBP-51, is known to inhibit GR transcriptional
activity. The aim of the present work was to investigate the
concentration of the immunophilin FKBP-51 in the hippocampus and frontal cortex in a neurodevelopmental animal model
of schizophrenia. This model is based on administration of lipopolysaccharide to pregnant rats (in the second and third week
of pregnancy). Amount of FKBP-51 was measured by Western
blot method. In order to verify the above model, exploration,
efficacy of sensorimotor gating and performance in the social interaction test were determined. Prenatal LPS treatment induced
behavioral disturbances typical of schizophrenia, like sensorimotor gating deficit, higher exploratory activity and changes
in social interaction test in the adult offspring. Furthermore, the
level of the immunophilin FKBP51 was lower in both female and
male offspring. These results suggest that multiple administrations of LPS to pregnant rats evoke GR hyperfunction in adult
offspring by decreasing the concentration of FKBP-51, a protein
which is known to inhibit GR function. This study was partially
supported by the grant N40101231/0174 from the MSHE, Warsaw, Poland.
TII.08
NUCLEAR β-CATENIN IS CONSTITUTIVELY PRESENT
IN THALAMIC NEURONS
Misztal K.1, Wisniewska M.1, Klejman M.1, Filipkowski R.K.2,
Kuznicki J.1
1
Laboratory of Neurodegeneration, International Institute of
Molecular and Cell Biology, Warszawa, Poland; 2 Laboratory of
Molecular Neurobiology, Nencki Institute of Experimental Biology
TII.10
IMMUNOHISTOCHEMICAL ANALYSIS OF
NEUROPEPTIDES DISTRIBUTION IN SPODOPTERA
EXIGUA AND PYRRHOCORIS APTERUS BRAIN
Bembenek J.1, Bugla K.1, Gladysz M.1, Francikowski J.1,
Takeda M.2
1
Department of Animal Physiology and Ecotoxycology, University
of Silesia, Katowice, Poland; 2 Graduate School of Science and
Technology, Kobe University, Kobe, Japan
Wnt signaling is one of the most important mechanisms engaged in embryonic development. In the adult organism it is present in regenerating
tissues. The key mediator in the signaling is β-catenin, which translocates
to the nucleus and triggers transcription of the Lef1/Tcf target genes.
Recently, expression of some of the Wnt pathway components has been
observed in the thalamus of the adult brain. We show that β-catenin and
Lef1 are constitutively present in nuclei of thalamic neurons in vivo. We
also demonstrate that β-catenin accumulates in about 40% of thalamic
cells in vitro, without any prior stimulation. This phenomenon does not
depend on soluble factors produced by glia cells or cortical neurons,
because nor glia cells co-culture neither cortical conditioned medium
affects the number of β-catenin positive cells. These observations support a new idea that Wnt/β-catenin signaling is an intrinsic feature of
thalamic neurons, independent on thalamo-cortical interaction what
was proposed before. Whether the nuclear localization of β-catenin in
thalamic neurons is a consequence of autocrine Wnt stimulation or a
specific regulation inside the cells remains to be determined.
The corazonin (Crz), was originally purified as a cardioacceleratory
neuropeptide in P. americana. Following studies indicated that Crz
induces dark colorization and morphometric changes in locust; reduces the spanning rate of silk in B. mori; is implicated in circadian
clock. The pigment dispersing hormone/pigment dispersing factor
(PDH/PDF), primary was described in crustaceans and in following studies in insects. Researchers have shown its role in circadian
organization of insects as a important output factor of circadian
clock. CCAP (crustacean cardioactive peptide), has a broad range
of activity including: cardioacceleratory effect; affecting muscle
and gut functions; induces release of AKH. Mapping of Crz-, PDF-,
CCAP- containing neurons provides an important information concerning sites of release and action of these peptides. Here we employed immunohistochemical methods to confirm the presence of
above mentioned neuropeptides and to investigate their distribution
in the brain of Spodoptera exigua (Lepidoptera) and Pyrrhocoris
apterus (Hemiptera). Using [His7]-Crz antibody we were able to
detect immunoreactive signal in both species in dorsolateral (DL)
neurosecretory cells projecting ipsilateraly to the corpora cardiaca.
Additional immunoreative somata were observed in pars intercerebralis (PIC). PDF-like immunoreativity was observed in optic lobes,
DL and much weaker in PIC. CCAP-ir was detected in OL and PIC.
TII.09
EXPRESSION REGULATION OF A NOVEL
Ca2+ BINDING PROTEIN CALMYRIN 2 (Cib2)
INDICATES ITS FUNCTION IN NMDA RECEPTOR
MEDIATED Ca2+ SIGNALING
Debowska K.1, Blazejczyk M.1, Sobczak A.1, Jaworski J.2,
Kuznicki J.1, Wojda U.1
1
Laboratory of Neurodegeneration, 2 Laboratory of Molecular and
Cellular Neurobiology, International Institute of Molecular and Cell
Biology, Warszawa, Poland
Calmyrin 2 (CaMy2, Cib2) represents a novel calmyrin subfamily of Ca2+ binding proteins that coordinate Ca2+ in the EF-hand motives. CaMy2 mRNA was recently detected in brain, but nothing is
known about CaMy2 protein localization and properties in the brain.
We have cloned CaMy2 from rat brain, demonstrated CaMy2 Ca2+sensor properties and determined its neuronal pattern of rat brain expression. CaMy2 protein is expressed mainly in hippocampal neurons
and localizes together with Golgi apparatus and dendrite markers. Our
studies in primary cultures of hippocampal neurons demonstrate that
expression of CaMy2 protein is induced upon neuronal activation with
pharmacological agents that stimulate Ca2+ influx through such types
of Ca2+ channels as glutamate excitatory receptors and voltage-operated Ca2+ channels. In addition, increase in CaMy2 protein level was
induced by soluble amyloid β and BDNF. However, most prominent
increase in CaMy2 protein (7-fold), and mRNA (2-fold) occurs upon
stimulation of NMDA receptor (NMDAR). The induction is blocked
by translation inhibitors, specific antagonists of NMDAR, the Ca2+-chelator BAPTA, and inhibitors of ERK1/2 and PKC, kinases transmitting NMDAR-linked Ca2+ signal. Our results show that CaMy2 level
is controlled by NMDAR and Ca2+ and suggest CaMy2 role in Ca2+
signaling underlying NMDAR activation. This work was supported by
the Polish Ministerial Research grant N301 109 32/3854.
TII.11
GENERATION OF TRANSGENIC MICE WITH
SELECTIVE ABLATION OF NMDA RECEPTORS
IN NORADRENERGIC NEURONS
Rodriguez Parkitna J.1, Tokarski K.2, Golembiowska K.3,
Kubik J.1, Solecki W.1, Novak M.4, Schütz G.4, Przewlocki R.1
1
Department of Molecular Neuropharmacology, 2 Department of
Physiology, 3 Department of Pharmacology, Institute of Pharmacology
PAS, Kraków, Poland; 4 Molecular Biology of the Cell I, German
Cancer Research Center, Heidelberg, Germany
The brain’s noradrenergic system system provides essential modulation to neuronal activity, with well established roles in control of
arousal and selective attention, as well as memory, learning and response to stress. We have generated a transgenic mouse, NR1DbhCre,
with ablation of the essential NMDA receptor subunit NR1 (Grin1)
in noradrenergic cells. Since no functional NMDA receptors may
be formed in the absence of NR1, and their expression is restricted to the central nervous system, the NR1DbhCre mice have impaired
glutamate-dependent plasticity in the central noradrenergic neurons, without observable alterations in the sympathetic system or
the hypothalamus-pituitary-adrenal axis. Transgenic animals were
born at expected ratios and developed normally, displaying no obvious impairments. The general anatomy of the noradrenergic system in the mutant mice was normal, no loss of cells was observed
and noradrenaline content in the prefrontal cortex was not altered.
Interestingly, preliminary electrophysiological analysis indicates
300
that loss of functional NMDA receptors attenuates the spontaneous
activity in current-clamped locus coeruleus noradrenergic neurons
manually held at −50 mV potential. In summary the NR1DbhCre transgenic mice are a novel model for the study of the roles of the noradrenergic system in the central nervous system.
TII.12
THE EFFECT OF BIOGENIC AMINES SEROTONIN,
DOPAMINE, OCTOPAMINE AND TYRAMINE ON
AGGRESSIVE BEHAVIOUR OF WORKERS OF
THE RED WOOD ANT FORMICA POLYCTENA
Szczuka A. 1, Wnuk A. 1, Korczyńska J. 1, Symonowicz B. 1,
Gonzalez Szwacka A. 1, Mazurkiewicz P. 2, Francikowski J. 3,
Mirecka A. 1, Kostowski W. 4, Godzińska E. J. 1
1
Laboratory of Ethology, Department of Neurophysiology, Nencki
Institute of Experimental Biology PAS, Warszawa, Poland; 2 College
of Inter-Faculty Individual Studies in Mathematics and Natural
Sciences, Warsaw University, Warszawa, Poland; 3 Department
of Animal Physiology and Ecotoxicology, University of Silesia,
Katowice, Poland; 4 Department of Pharmacology and Physiology
of the Nervous System, Institute of Psychiatry and Neurology,
Warszawa, Poland
We investigated the effect of abdominal injections of 4 biogenic
amines – serotonin (5-HT), dopamine (DA), octopamine (OA) and
tyramine (TA) – on behaviour of workers of the red wood ant
Formica polyctena in 4 types of aggression tests. The ants were
observed during a 10 min encounter with a nestmate, a conspecific ant from an alien colony, a worker of another ant species
(Formica fusca), and a small larva of the house cricket (Acheta
domesticus). DA administration had the most important effect on
ant aggressive behaviour. DA injections exerted a stimulatory effect on threats directed to F. fusca and to larvae of A. domesticus
and on biting behaviour directed to larvae of A. domesticus. TA
suppressed the frequency and the duration of biting behaviour directed to these larvae, although that effect was significant only
when the effects of TA were compared with those of DA and 5-HT.
TA also reduced self-grooming behaviour, but only in tests with
alien conspecifics. Other effects of biogenic amine administration included a stimulatory effect of OA on other active behaviour
displayed by the ants during the tests with A. domesticus, inhibitory effects of 5-HT, OA and TA on allogrooming of a nestmate,
and lenghtening of the latency to the first antennal contact with
a nestmate observed in TA and 5-HT-treated ants. The most important effects of biogenic amine administration were observed in
the case of interactions of the ants with crickets. Support: grant
MNiSW N N303 3075 33.
TII.13
CALCIUM-BINDING PROTEINS IN THE
HIPPOCAMPAL FORMATION OF THE GUINEA PIG
Kolenkiewicz M., Robak A.
Department of Comparative Anatomy, University of Warmia
and Mazury in Olsztyn, Olsztyn, Poland
Calcium ions (Ca 2 ) are known as a second messenger in neurons. However, the concentration of Ca 2 should be maintained
at an optimal level in view of their high toxicity. Calcium-binding proteins (CaBPs) bind Ca 2 with high affi nity. The amount
of the CaBPs is age-related and varies during brain development. The aim of this study was to analyze the distribution of
CaBPs, parvalbumin (PV) and calbindin D-28k (CB), in the
hippocampal formation (the dentate gyrus – DG and hippocampus proper – HP). The studies were carried out on the brains
of newborn (P0) and 80-day-old (P80) guinea pigs. Labelling
immunofluorescence was performed on 10 μm-thick frozen
sections. PV- and CB-immunoreactive (PV-IR, CB-IR) structures were identified with antibodies against PV (1:2000, code
P3088) and CB (1:2000, code C9848). Both studied parts are
composed of three cellular layers. In the dentate gyrus, PV-IR
cells (elongated and oval-shaped) were labelled in the hilus. In
the middle (granular) layer only PV-IR dots surrounding negative cells were observed, whereas in the third (molecular) layer
no immunoreactive structures were seen. In the hippocampus
proper PV-IR neurons (triangular, piriform and oval-shaped)
were found in each sector (CA1–CA3) of the pyramidal layer,
the same as in the oriens and molecular layers. CB-IR neurons
(oval-shaped, densely arranged) were observed mainly in the
granular and pyramidal layers. The distribution of the studied
calcium-binding proteins was similar in P0 and P80.
TII.14
REGULATION OF SERUM RESPONSE FACTOR-DRIVEN
TRANSCRIPTION BY MKL2 (MEGAKARYOBLASTIC
LEUKEMIA 2) IN RESPONSE TO BDNF
Kalita K.1, Rejmak E.1, Kaczmarek L.1, Hetman M.2
1
Department of Molecular and Cellular Neurobiology, Nencki
Institute of Experimental Biology PAS, Warszawa, Poland;
2
Kentucky Spinal Cord Injury Research Center and Department of
Neurological Surgery, University of Louisville, Louisville, KY, USA
SRF-mediated transcription contributes to brain plasticity.
However, it is unclear which of the several SRF co-activators
participates in transcriptional events underlying the formation
and modifications of neuronal circuitries. We investigated the
role of SRF co-activator, MKL2, in regulation of SRF-driven
transcription in neurons. MKL2 expression was observed in
newborn cortical or hippocampal neurons in culture as well as
in adult rat forebrain. In-situ hybridization showed the presence
of MKL2 mRNA in all fields of the hippocampus, especially in
dentate gyrus. Neither overexpression nor inhibition of MKL2 by
shRNA caused apoptotic cell death in neurons. Overexpression
of MKL2 in primary cortical neurons enhanced SRF-driven
transcription elevated by BDNF stimulation. In addition, inhibition of MKL2 reduced BDNF activation of SRF-driven
transcription, on classical SRE promoters. MKL2 is a less
potent activator of SRF-mediated transcription than recently
studied member from the same family of co-activators MKL1.
Interestingly, MKL2 is working as a partial inhibitor of BDNF
activated MKL1-dependent transcription in case of promoters with isolated SRF binding sites. These results suggest that
MKL2 contributes to BDNF-mediated regulation of SRF-driven gene expression. Particular mode of regulation depends on
the presence of a second co-activator MKL1 or the type of SRF
binding sites within the regulatory regions of respective genes.
FNP Homing - MF EOG to KK, RO1 (NS047341-01) to MH.
TII.15
ADMINISTRATION OF CLENBUTEROL IMPROVES
MORPHOLOGY OF CHOLINERGIC NEURONS AND
COGNITIVE PROCESSES IN AGED RATS
Mazurkiewicz M.1, Mietelska-Porowska A.1, Robakiewicz I.1,
Gasiorowska A.2, Baksalerska-Pazera M.1, Niewiadomska G.1
1
Biology PAS, Warszawa, Poland; 2 Department of Applied Physiology,
genic animals displayed impaired object recognition memory, but
increased performance in right/left discrimination test. In addition, the NR1DbhCre mice displayed higher food reward sensitivity
as evidenced by faster acquisition of food self-administration and
higher breaking point in progressive ratio schedule of reinforcement. In conclusion, the ablation of NMDA receptors in noradrenergic neurons in mice produce impairment of recognition memory
and alteration of motivational systems.
A deficit in the cholinergic system of the basal forebrain (BF) is
thought to contribute to the development of cognitive symptoms of
dementia. Forebrain cholinergic neurons are highly dependent on
nerve growth factor (NGF) for phenotype maintenance. Attempts
to prevent age-associated cholinergic vulnerability and deterioration therefore represent a crucial point for pharmacotherapy in the
elderly. The pharmacological induction of endogenous NGF synthesis in the brain could be an elegant way to overcome application
problems. Therefore, the present experiment was undertaken to
determine the influence of prolonged pharmacological stimulation of NGF biosynthesis on learning and memory in aged rats.
To address these issues we used young (4-month-old) and aged
(28-month-old) rats in which we stimulated endogenous NGF biosynthesis by treatment with clenbuterol (β2-adrenergic receptors
agonist). The cognitive behavior of the young and aged rats was
assessed in the long-lasting “Non-Matching to Position Test”. Our
data suggest that in aged rats, clenbuterol positively affects cognitive processes related to formation of associations established in
recognition memory and discrimination tasks. In addition, NGF
significantly improved morphological parameters of BF cholinergic cells in aged rats. These data suggest that endogenous NGF
induction restores the age-related decline of the cholinergic system activity in rats, resulting in improvement of water maze performance in old animals.
TII.17
DISTRIBUTION OF CAPACITATIVE CALCIUM ENTRY
ACTIVATOR STIM1 IN THE MOUSE BRAIN
Skibinska-Kijek A., Kuznicki J.
Laboratory of Neurodegeneration, International Institute of Molecular
and Cell Biology, Warszawa, Poland
TII.16
ANXIETY, DEPRESSIVE, COGNITIVE AND
MOTIVATIONAL PHENOTYPE IN MICE WITH
SELECTIVE ABLATION OF NMDA RECEPTORS
IN NORADRENERGIC NEURONS
Solecki W.1, Rodriguez Parkitna J.1, Kubik J.1, Schütz G.2,
Przewlocki R.1
1
Department of Molecular Neuropharmacology, Institute of
Pharmacology PAS, Kraków, Poland; 2 Molecular Biology
of the Cell I, German Cancer Research Center, Heidelberg, Germany
The noradrenergic system of the brain has been postulated to mediate arousal, selective attention, as well as anxiety, depression
and learning and memory. Here we report the behavioral phenotype of transgenic mouse (NR1DbhCre) with selective ablation of the
NMDA receptor subunit NR1 in noradrenergic cells. We focused
on behaviors modulated by the noradrenergic system, in particular anxiety-like behaviors in the light/dark box, and open field as
well as depressive-like behaviors in forced swim test. Furthermore, memory performance was measured using elevated plus
maze spatial learning (EPMSL), Y maze, right/left discrimination, habituation to open filed and novel object recognition and
motivation-dependent behaviors was studied using food pellet
self-administration paradigm. While no alterations were found in
anxiety- and depressive-like behaviors in NR1DbhCre mice, the trans-
Capacitative calcium entry (CCE) is a significant component of
calcium homeostasis in non-excitable cells. In neurons, an increasing number of evidence points to CCE as an important event in
neuronal physiology and pathology. STIM1 is an endoplasmic reticulum (ER) residing protein, where it serves as a calcium sensor.
Low level of calcium in ER drives STIM1 oligomerize and interact
with plasma membrane protein Orai1, hence leading to calcium
entry. Previously, we showed STIM1 expression in neurons, where
it exhibits the same mode of action as described for non-excitable
cells. Here we present description of STIM1 distribution in the
mouse brain. The highest STIM1 immunoreactivity was observed
in Purkinje neurons and their dendrites. Very high immunoreactivity was found in the basal ganglia. High immunoreactivity was
present in the hippocampal formation, in the piriform cortex, also
in infragranular layers and layer V pyramidal neurons in the neocortex. In amygdala most nuclei showed strong immunoreactivity,
only the basolateral complex was weakly stained. The thalamus
was very weakly stained, and the hypothalamus showed immunoreactivity of both cells and neuropil. Observed differences in
STIM1 distribution in the brain indicates that various brain structures depends on CCE to different extend. Prominent dissimilarity
of STIM1 immunoreactivity between amygdaloid nuclei is, in our
opinion, the most interesting in light of anatomical and functional
organization of the amygdala.
TII.18
PROTEIN SUMOYLATION IN SYNAPTOSOMES,
EFFECTS ON GLUTAMATE RELEASE AND
PRESYNAPTIC Ca2+ INFUX
Feligioni M.
Department of Anatomy, University of Bristol, Bristol, UK
SUMO-1 is a protein with similar structure to ubiquitin. SUMOylation modifies proteins structure effecting their molecular
mechanisms. SUMO-1 is present in neurons and in the synapse.
Here we are investigating whether SUMO-1 is a presynaptic
protein and whether SUMOylation modulates glutamate release.
Ultrasynaptic separation approach was used to investigate the
distribution of SUMO-1 in peri-, post- and presynaptic compartments in synaptosomes. Presynaptic protein SUMOylation is
augmented by KCl or AMPA stimulus and is decreased by kainate. NMDA has no influence presynaptic protein SUMOylation
level. Synaptosomes glutamate release was measured. Recom-
302
binant SUMO-1 and SENP-1, a SUMO-specific proteases which
cleave SUMO from its substrates, where used with their mutated
recombinant variant in synaptosomes as entrapped proteins. KCl
stimulated glutamate release is increased when synaptosomes
contain SENP-1 but is decreased when synaptosomes contain
full-length SUMO-1. It was observed decrease in kainate evoked
glutamate release from synaptosomes preloaded with SENP-1
but a marked increase in release from synaptosomes preloaded
with SUMO-1. Intracellular Ca 2+ concentration was also measured. Wild-type SENP-1 increased Ca 2+ influx evoked by KCl
or AMPA and decreased it following a kainate challenge. Conjugatable SUMO-1(GG), on the other hand, reduced Ca 2+ influx
evoked by KCl or AMPA and increased the levels of Ca 2+ influx
elicited by kainate
TII.19
THE EFFECT OF PROLONGED ZOLPIDEM
TREATMENT ON GABA A RECEPTORS IN PRIMARY
CULTURE OF RAT CEREBELAR GRANULE NEURONS
Vlainic J., Pericic D.
Department of Molecular Medicine, Rudjer Boskovic Institute,
Zagreb, Croatia
The aim of this study was to further explore the mechanisms
leading to adaptive changes in GABA A receptors following their
prolonged exposure to hypnotic zolpidem, a positive allosteric
modulator of GABA A receptors. Imidazopyridine zolpidem is the
most widely prescribed non-benzodiazepine hypnotic, with preferential, although not exclusive, binding for receptors containing
α1 subunit. It was suggested that drugs with high selectivity for
α1 containing receptors produce upon repeated treatment less
tolerance and dependence than classical benzodiazepines. As an
extension of our previous work, we treated cerebellar neuronal
cells isolated from 8-days old rats with 10 μM zolpidem during
48 h. Radioligand binding studies and RT-PCR analysis were
preformed under conditions previously described [Pericic et al.
(2007) Naunyn Schmiedebergs Arch Pharmacol 375: 177–187].
Results demonstrate that prolonged treatment of these cells with
zolpidem induced changes neither in GABA A receptor number nor
in expression of α1 subunit mRNA. As evidenced by a decreased
ability of GABA to stimulate [3H]flunitrazepam binding, chronic
exposure of these cells to zolpidem produced the functional uncoupling between GABA and benzodiazepine binding sites on
GABA A receptor complex. If this mechanism is responsible for
the development of tolerance following chronic administration of
classic benzodiazepines, than one might can expect that chronic
zolpidem might also induce tolerance.
TII.20
MODULATION OF NEURONAL MEMBRANE
PROPERTIES BY THE COXSACKIEVIRUSADENOVIRUS RECEPTOR
Cholewa J., Juettner R., Rathjen F.
Department of Developmental Neurobiology, Max-Delbrueck-Center
for Molecular Medicine, Berlin, Germany
The Coxackievirus and Adenovirus receptor (CAR) was originally identified as a cell surface protein enabling both viruses
to interact with cells. Besides this pathological role as a vi-
rus receptor, the physiological function of CAR in the CNS
is largely unknown. CAR is a member of the Ig superfamily
composed of two Ig-like domains, a transmembrane stretch
and a cytoplasmic segment. CAR is expressed early in development on neurons and becomes restricted to synapse-rich layers
at more advanced stages. Application of the fiber knob of the
adenovirus, which binds to CAR, resulted in longer neurites
in comparison to the untreated cell cultures. Furthermore, the
formation of cell aggregates was reduced by the fiber knob. By
using whole-cell patch clamp technique we analyzed the effect
of the fiber knob on passive membrane properties and synaptic
activity. The fiber knob reduces the membrane resistance (Rm)
of cultured neurons. Consistently, in CAR deficient neurons
Rm was significantly higher compared to wild type neurons,
and application of fiber knob had no effect on Rm on CARdeficient neurons. Thus, CAR may influence Rm via membrane
proteins with a membrane conductance (e.g. ion channels, gap
junction). Our model is, that CAR deficient neurons may not
express or express some not functional ion channel or transport
proteins in the membrane. On the other side, binding of the
fiber knob to CAR might induce activation – opening of such
transport protein.
TII.21
CULTURE CONDITIONS SEVERELY ALTER THE
PROPORTIONS BETWEEN GABA- AND GLUTAMATEEVOKED CURRENTS IN HIPPOCAMPAL PYRAMIDAL
NEURONS
Szczot M., Wojtowicz T., Mozrzymas J.
Department of Biophysics, Laboratory of Neuroscience, Wroclaw
Medical University,Wrocław, Poland
Cultured hippocampal cells are commonly used as a convenient
model but non-physiological conditions (impaired homeostasis,
lack of glial cells etc.) persisting over long period of time raise
concerns. Acute brain slices are believed to better maintain
the physiological features but are often problematic for technical reasons (e.g. complicated access to neurons, attenuated
spread of pharmaceutics). Non-physiological conditions in cell
cultures during maturation of GABAergic and glutamatergic
systems may alter expression of GABA A and glutamate receptors affecting thus the excitation to inhibition balance. Proportion between GABA and glutamate receptors may be evaluated
by determining ratio between amplitudes of current responses
to saturating agonist concentrations. We have recorded current
responses to ultrafast applications of saturating GABA and glutamate concentrations in hippocampal cell cultures (9–15DIV)
and in brain slices from CA1 pyramidal neurons of P19–P23
rats. For cell cultures, GABAergic and glutamergic currents
ratio was 2.34 ± 0.45 (n=12) while for the brain slices it was
only 0.24 ± 0.02 (n=5). These results provide evidence that
non-physiological conditions in cell cultures may cause a dramatic change in expression pattern of GABA A and glutamate
receptors. The underlying mechanisms are not known, but we
may hypothesize that a homeostatic modulation due accumulation of neurotransmitters in culture medium could be involved.
Support: grant no. 070231/Z/03/Z.
TII.22
MATRIX METALLOPROTEINASE-9 INCREASES NMDA
RECEPTOR LATERAL DIFFUSION
Michaluk P.1, Mikasova L.2, Groc L.2, Frischknecht R.3,
Choquet D.2, Kaczmarek L.1
1
2
Laboratory of Cellular Physiology of the Synapse, University of
Bordeaux 2, Bordeaux, France; 3 Department of Neurochemistry
and Molecular Biology, Leibniz Institute for Neurobiology,
Magdeburg, Germany
Matrix metalloproteinase-9 (MMP-9) has emerged as a physiological
regulator of NMDA receptor (NMDAR)-dependent synaptic plasticity and memory. The pathways by which MMP-9 affects NMDAR
signaling remain, however, not well understood. Using single Quantum Dot tracking we demonstrate that MMP-9 enzymatic activity
increases NR1-NMDAR surface trafficking but has no influence on
AMPA receptor (AMPAR) mobility. Other extracellular protease –
Cathepsin G has no effect on both NMDAR and AMPAR lateral diffusion. The mechanism of MMP-9 action on NMDAR is caused neither by change in overall extracellular matrix (ECM) structure, nor
by cleavage of NMDAR subunits, but by influence on integrinbeta 1
signaling. These findings describe a new target pathway for MMP-9
action in key physiological and pathological brain processes.
TII.23
INVOLVEMENT OF GROUP I METABOTROPIC
GLUTAMATE RECEPTORS IN FORMING HYPOXIC
BRAIN TOLERANCE INDUCED IN RATS BY
HYPOBARIC PRECONDITIONING
Belyakov A.1, Semenov D.1, Tjulkova E.1, Glushchenko T.1,
Salinska E.2, Samoilov M.1, Lazarewicz J.2
1
Laboratory of Regulation of Brain Neurons Function, Pavlov
Institute of Physiology RAS, Saint Petersburg, Russia; 2 Department
of Neurochemistry, Mossakowski Medical Research Centre PAS,
Warszawa, Poland
The assumption that hypoxic tolerance induced by preconditioning
with moderate hypobaric hypoxia (MHH) includes modification of intracellular Ca2+ (Ca2+i) transients by modulation of glutamate receptors
(GluR) activity was tested. Superfused rat brain slices were prepared 24
hours after three MHH sessions equivalent to 5 000 m above see level
and tested by application of selective agonists of different subtypes of
GluR. Ca2+i level in response to the agonists was increased in comparison with the control ones. The most prominent Ca2+ level shifts were
recorded in response to stimulation of group I metabotropic glutamate
receptors (ImGluRs). The modified pattern of the responses showed
striking prevalence of signal pathways responsible for Ca2+ release from
intracellular stores against modulation of Ca2+ entry. The selective antagonism of both 1 and 5 subtypes of ImGluRs in vivo during MHH did
not suppress the preconditioning efficiency, tested in the slices by severe (10 min) anoxia 24 hours after. Moreover by immunocytochemical
and Western-blotting methods we found no valid distinctions in a peptide expression of these ImGluRs subtypes between control and MHHpreconditiond rats. Thus it can be supposed that the changed pattern of
Ca2+ response to the agonist and mechanisms of brain tolerance induced
by MHH are determined by a shift in balance of different glutamatergic
signal pathways controlling the dynamics of Ca2+i level rather than by
modification of the agonist reception.
TII.24
GLUTAMATE AND NEUROPEPTIDE RELEASED
IN THE BRAIN OF MALE GRASSHOPPER
CHORTHIPPUS B. ALTER THE CONTEXT OF THE
PRODUCED SINGING IN A STEP-WISE MANNER
Vezenkov S.
Department of Logopedics, South West University Neofit Rilski,
Blagoevrad, Bulgaria
Injections of neuropeptide proctolin into brain can elicit singing
behaviour in male grasshoppers of the species Ch.biguttulus. It has
been shown earlier that activation of mAChRs lead to prolonged
calling song – first level of excitation. Proctolin release might
be associated with courtship singing and could probably trigger
switches between calling and courtship singing when a con-specific female responds acoustically – second level of excitation. Coinjections of proctolin with glutamate could alter the time course
of proctolin-stimulated singing by appearance of precopulatory
movements or led to appearance of additional behaviours such as
wing flappings – third level of excitation. The singing patterns are
produced only by the hind legs stridulatory movements rubbing
to the wing’s vein and driven by internal neuronal oscillator. The
context of the acoustic patterns depends only on the time courses
of these hind legs’ movements. The arousal of excitation was not
linear but in a step-wise manner alternating the described patterns
above. These pattern alterations were triggered by co-release of
few neurotransmitters – ACh, proctolin and glutamate. There
were indications that the intracellular signalling pathways coupled
to mAChRs, Proctolin receptors and mGluRs interact each other
to achieve the switches between the particular oscillator activity.
The latter might alternate the outgoing message context, produced
by the male in a given communicative situation.
TII.25
FACILITATION OF TRANSMITTER RELEASE FROM
RAT SYMPATHETIC NEURONS VIA PRESYNAPTIC
P2Y1 RECEPTORS
Chandaka G., Boehm S., Drobny H.
Department of Pharmacology, Medical University of Vienna, Wien,
Austria
P2 receptors for purines and pyrimidines are divided into 7 P2X
and 8 P2Y receptors (Abbracchio et al. 2006). Noradrenaline (NA)
and ATP are the predominant neurotransmitters in the sympathetic
nervous system. In rat superior cervical ganglion (SCG) neurons,
endogenous ATP activates presynaptic P2X receptors and thereby
mediates a positive feedback regulation of NA release (Boehm 1999).
In addition, endogenous ADP mediates a feedback inhibition of NA
release, through an inhibition of voltage activated calcium channels (VACCs) most likely via P2Y12 and pertussis toxin-sensitive G
proteins (Lechner et al. 2004). In sympathetic neurons, receptors
coupled to Gq type G proteins and PLC, such as B2 bradykinin and
M1 muscarinic receptors, mediate an inhibition of KCNQ channels
(Delmas et al. 2005). We found out that presynaptic P2Y1 receptors
mediate a facilitation of transmitter release from sympathetic nerve
terminals via PLC. The present study provides the first evidence for
a facilitatory P2Y receptor, in sympathetic neurons. Since P2Y1 antagonists are being developed as antithrombotics, this mechanism
can be expected to contribute to their pharmacodynamic profile, in
particular with respect to the vascular tone.
304
TII.26
MONOAMINES EFFECTS ON DIFFERENT TYPES OF
HIPPOCAMPAL GAMMA OSCILLATIONS IN ADULT
RAT IN VITRO
Wojtowicz A.1, van den Boom L.1, Chakrabarty A.1, Maggio N.2,
ul Haq R.1, Behrens C.1, Heinemann U.1
1
Institute of Neurophysiology, Johannes Müller Center of Physiology,
Charité, Berlin, Germany; 2 Department of Neurobiology, The
Weizmann Institute, Rehovot, Israel
Monoamines are implicated in cognitive processes in variety of
brain regions including the hippocampal formation, where storage and retrieval of information is facilitated by synchronous
network activities. We have investigated the effects of norepinephrine, serotonin and dopamine on carbachol-, kainate- and
stimulus-induced hippocampal γ-oscillations employing extra
and intracellular recordings. Monoamines dose-dependently and reversibly suppressed kainate- and carbachol-induced
γ-oscillations. The effect of serotonin was mimicked by fenfluramine, which releases serotonin from presynaptic terminals. Forskolin also suppressed kainate- and carbachol-induced
γ-oscillations. This effect was mimicked by 8-Br-cAMP and
isoproteronol, an agonist of noradrenergic β-receptor suggesting
that the monoamines-mediated suppression of these oscillations
could involve intracellular cyclic AMP. By contrast, stimulus-induced γ-oscillations were dose-dependently augmented in power
and duration after monoamines application. Intracellular recordings from pyramidal cells revealed that monoamines prolonged
the stimulus-induced depolarization and membrane potential oscillations. Stimulus-induced γ-oscillations were also suppressed
by isoproteronol, the D1 agonist SKF-38393 forskolin and 8-BrcAMP. This suggests that the augmentation of stimulus induced
γ-oscillations by monoamines involves – at least in part – different classes of cells than in case of carbachol- and kainate-induced
γ-oscillations.
TII.27
LEF1/BETA-CATENIN REGULATES Cav3.1 T-TYPE
CALCIUM CHANNEL GENE (CACNA1G) IN THE
THALAMUS
Wisniewska M.1, Michowski W.1, Lesniak W.2, Misztal K.1,
Dabrowski M. 3, Kuznicki J.1
1
Laboratory of Neurodegeneration, International Institute of
Molecular and Cell Biology, Warszawa, Poland; 2 Department of
Molecular and Cell Neurobiology, 3 Department of Cell Biology,
Nencki Institute of Experimental Biology PAS, Warszawa, Poland
Lef1/Tcf transcription factors together with β-catenin activate genes involved in proliferation and differentiation
of neuronal precursor cells. In mature neurons β-catenin
participates in dendritogenesis and synaptic function as a
component of the cadherin cell-adhesion complex, its transcriptional activity, however, remains elusive. We observed
that in the adult brain Lef1 and β-catenin accumulate specifically in the nuclei of thalamic neurons, implying their involvement in regulating gene expression in these cells. The
electrophysiological properties of thalamic cells depend on
T-type low-voltage-gated Ca 2+ channels. Since Cav3.1 is the predominant T-type pore subunit in the thalamus, we hypothesized
that the Lef1/β-catenin complex regulates transcription of its
gene. The increase of Cav3.1 gene expression in thalamic cells
treated in vitro with Wnt3A, an activator of β-catenin, corroborated our presumption. Analysis of the Cav3.1 gene promoter
revealed that its proximal region contains a Lef1 binding site.
Indeed, luciferase assays confi rm that the promoter is activated by Lef1 and β-catenin. Furthermore, chromatin immunoprecipitation demonstrated that the promoter is occupied by
β-catenin in the thalamus. Together, our data indicate that the
Lef1/β-catenin complex regulates transcription of Cav3.1 gene.
We propose that β-catenin contributes to neuronal excitability
not only by a local action at the synapse, but also by activating
gene expression.
TII.28
CYCLICAL EXPRESSION OF THE SODIUM PUMP
α-SUBUNIT GENE AND PROTEIN IN THE VISUAL
SYSTEM OF DROSOPHILA MELANOGASTER
Krzeptowski W., Gorska-Andrzejak J., Pyza E.
Department of Cytology and Histology, Jagiellonian University,
Kraków, Poland
In the lamina L1 and L2 monopolar cells show circadian
rhythms in shape and size changes, however, mechanisms of
these rhythms have not been recognized yet. It has been proposed that swelling and shrinking of L1 and L2 interneurons
might be due to ionic fluxes, changes in cytoskeleton organization and in protein synthesis. This process also depends on
glial cell activity. Since ion channels seem to be involved in
morphological plasticity of neurons and glial cells we examined expression of the sodium pump, Na+,K+-ATPase, during
the day and night. The sodium pump consists of two polypeptide chains: α- and β-subunits encoded by the Atpα and Nrv2
genes, respectively. Using the real-time PCR and immunohistochemistry methods we found the α-subunit shows daily
changes in mRNA and protein concentrations while in case the
β-subunit such changes were not observed. Since the rhythm in
the α-subunit abundance was not observed in the per 0 arrhythmic mutant, this rhythm is controlled by clock genes. It seems
to reflect changes in the visual system activity.
TII.29
ROLE OF THE FAST VOLTAGE GATED K+ CHANNEL
FUNCTION IN FIRING BEHAVIOUR OF PURKINJE
NEURONES OF ATAXIC RATS
Janahmadi M., Goudarzi I., Kaffashian M.
Department of Physiology, Neuroscience Research Center, Faculty
of Medicine, Shahid Beheshti University (Medical Campus), Tehran,
Iran
The role of fast inactivating (IA) voltage gated K+ channel function in shaping the fi ring activity of Purkinje cells (PCs) in ataxic rats was investigated. Ataxia was induced in 5 week-old male
rats of Sprauge-Dawley by single injection of 3-acethylpyridine
(65 mg/kg, i.p.). Four days following induction of ataxia, etheranesthetized rats were decapitated; brains rapidly removed and
300 μm thick parasagittal slices were prepared from the vermis of the cerebellum. Whole cell patch clamp recording was
achieved in the presence of synaptic blockers. To prevent the
induction of ataxia, 4-AP (2 mg/kg body weight), IA channel
blocker, was injected prior to 3-AP treatment. Results indicated
that in ataxic rats, the latency of the initial spike in PCs was significantly increased (P<0.001), while both fi ring frequency and
amplitude of Ca 2+ spikes were (P<0.001) suppressed compared to
control. However, pretreatment with 4-AP not only almost completely prevented the neuronal degeneration in PCs layer but also
the development of ataxia, which occurred in ataxic rats. PCs
in rats co-treated with 4-AP and 3-AP fi red predominantly in
rhythmic bursts. The amplitude of Ca 2+ spikes was significantly
(P<0.001) increased compared to ataxic rats. An increase in the
intrinsic excitability of PCs was also observed as a significant
decrease in the fi rst spike latency (P<0.001). In 4-AP co-treated rats, the duration of action potential was also significantly
lengthened (P<0.001).
TII.30
THE CONTRIBUTION OF NEURONAL GLUTAMATE
TRANSPORTERS IN SHAPING OF eEPSC IN CA1
HIPPOCAMPAL NEURONS
Kondratskaya E.1, Akaike N.2, Krishtal O.1
1
Department of Cellular Membranology, Bogomoletz Institute of
Physiology, Kiev, Ukraine; 2 Research Division for Life Sciences,
Kumamoto Health Science University, Kumamoto, Japan
Glutamate is the major excitatory transmitter in CNS although
it causes severe brain damage by pathologic excitotoxicity.
Central to efficient neurotransmission is a powerful protection afforded by specific high-affi nity glutamate transporters
in neurons and glia, clearing synaptic glutamate. In whole-cell
patch clamp experiments the influence of neuronal glutamate
transporters on spontaneous (sEPSC) and evoked postsynaptic
currents (eEPSC) in hippocampal CA1 neurons was examined
by manipulating the content of intracellular solution. With Cs+based internal solution the deficient of presynaptic glutamate
transporters affected the occurrence of synaptic event and thus
involved in the regulation of transmitter release. eEPSCs were
generally suppressed both in amplitude (to 48.73 % vs. control )
and in success rate (Rsuc ) by TBOA (10 μM) (from 91.1 % in
control to 79.57%). In contrast, with K+-based internal solution
(all GluT are intact), amplitude of eEPSC was substantially potentiated by pre-treatment with TBOA (up to 150%), whereas
(Rsuc) was reduced to 79.8% in average. The identical reduction
of event success rate as well as increased PPF ratio for eEPSC in
both cases is indicative for TBOA effect on presynaptic uptake
site. In conclusion, presynaptic transporters are suggested to act
mainly as negative feedback signal on presynaptic release and/
or referred to vesicle refilling processes, when postsynapticaly
located transporters are supposed to shape postsynaptic events.
TII.31
RECIPROCAL REGULATION OF KCC2 AND NKCC1 VIA
SPATIAL DISTRIBUTION IN THE MEMBRANE
Hartmann A.1, Wenz M.2, Kranz T.2, Eckhard F.2, Nothwang H.1
1
Department of Neurogenetics, Carl-von-Ossietzky University,
Oldenburg, Germany; 2 Animal Physiology Group, University of
Kaiserslautern, Kaiserslautern, Germany
Throughout the nervous system, the Cl--outward transporter
NKCC1 and the Cl--inward transporter KCC2 play an impor-
tant role in setting the intracellular chloride concentration.
Both transporters are co-expressed in many neurons. This
requires tight and reciprocal regulation, due to their opposite direction of Cl- transport. Here, we investigated the role
of cholesterol-rich membrane microdomains, so-called membrane rafts, for NKCC1 and KCC2 function. Membrane rafts
were isolated from mature rat brainstems at postnatal days (P)
25–30 via solubilization in Brij 58 followed by sucrose-density flotation experiments. This analysis revealed that NKCC1
was codistributed with the membrane raft marker flotillin-1
in lower density fractions. In contrast, two populations were
observed for KCC2. One was solubilized, whereas the other
population was detergent insoluble. Studies in HEK293 cells
demonstrated that disruption of membrane rafts by combined
treatment of methyl-b-cyclodextrin and sphingomyelinase impaired NKCC1 mediated 86Rb + uptake, whereas it increased
KCC2 dependent 86Rb + uptake. From these data we conclude
that NKCC1 requires association with membrane rafts for
transport activity, whereas KCC2 is inhibited by membrane
rafts. In agreement with this conclusion, transport inactive
KCC2 in the neonatal brainstem (P0–2) is exclusively present
in membrane rafts. Taken together, our analysis identifies a
cellular mechanism that might be involved in reciprocal regulation of NKCC1 and KCC2.
TII.32
GABA TRANSPORTER GAT-1-POSITIVE (GAT1+) PUNCTA MAY PLAY AN ACTIVE ROLE IN
MODULATING GABA ACTION IN THE “TRAINED
BARREL” CORTEX
Siucinska E.1, Pyza E.2, Jasinska M.2
1
Department of Molecular and Cellular Neurobiolology, Nencki
2
Department of Cytology and Histology, Institute of Zoology,
Jagiellonian University, Kraków, Poland
We have shown previously that classical conditioning training involving stimulation of a row of facial vibrissae (conditioning stimulus, CS) and electrical shock (unconditioned
stimulus, UCS) to the tail is associated with increase in the
numerical density of GAD67+ boutons located on the cognate
barrel hollows. The present study was designed to determine
whether the expression of GABA transporter GAT-1 changes
after associative learning using the mice classical conditioning training as a model. We found that numerical density of
GABA transporter GAT-1+ puncta was increased by about
50% in the neuropil of the ”trained barrel” hollow compared
with controls: pseudoconditioned, whisker stimulation without
UCS, and naive group of mice. The electron microscopic observations confi rm that immunoreactivity for GAT-1+ puncta
was localized: in single synaptic terminals present on symmetric specialization synapses, on double-synapse spines on
symmetric synapse, and on astrocytic processes. The projection of several confocal plans (3D spatial organization)
identified GAT-1+ coupled astrocytes. The present evidence
supports the hypothesis that GABA transporter GAT-1 localized on GABAergic terminals and the astrocyte networks
in the hollow of the “trained barrel” may be involved in
learning-dependent changes in layer IV of the barrel cortex.
Supported by MNiSW grant 188 to ES.
306
TII.33
QUANTAL ATP RELEASE FROM ACUTELY ISOLATED
CORTEX CELLS ON SINGLE BOUTON LEVEL
Palygin O., Pankratov Y.
Department of Biological Sciences, University of Warwick, Coventry,
UK
The ATP postsynaptic currents were measured during local extracellular electrical stimulation of one single bouton of acutely isolated cortex cells. The successful mediator fusion events were visually controlled by stimulus activating staining by SynaptoGreen.
The ATP was released from distinct vesicle population as evidence
by existence of two separate populations of synaptic currents. As
membrane potential was set above the reversal potential for Cl-, we
observed fast inward currents mediated by P2X receptors and slow
outward currents mediated by GABA. Furthermore, these two populations of synaptic currents we separated by pharmacological tools.
When slow outward postsynaptic currents we inhibited by picrotoxin, the residual fast inward currents were blocked by 30 μM PPADS.
The quantal parameters of postsynaptic currents in the cortical cells
were obtained and verified using maximum likelihood and autocorrelation techniques. The amplitude histograms for both mediators
have a binominal distribution character with quantal size near 6.5
pA for ATP and 10 pA for GABA. In a conclusion, our results demonstrate that release of ATP in cortical inhibitory terminals occurs
from separate pool of synaptic vesicles. Upon excitation of postsynaptic site release of ATP and GABA can be synchronized when both
transmitters are released in multivesicular package.
TII.34
KISSPEPTIN EFFECTS ON GABA TRANSMISSION
TO GONADOTROPIN-RELEASING-HORMONE
NEURONS ARE VIA BOTH PRE- AND POSTSYNAPTIC
MECHANISMS
Pielecka-Fortuna J., Moenter S.
Neuroscience Graduate Program, University of Virginia,
Charlottesville, VA, USA
Gonadotropin releasing hormone (GnRH) neurons control fertility
centrally. The neuromodulator kisspeptin (kiss) strongly activates
GnRH neurons via its G-protein coupled receptor 54 (GPR54). GnRH
neurons express GPR54, implicating direct actions of kiss on these
cells; however, GPR54 is broadly expressed in the brain, suggesting
indirect actions are possible. Estradiol potentiates the GnRH neuron
response to kiss, which recent work showed was due to kiss enhancement of excitatory GABA and glutamate transmission to GnRH neurons. Here we tested if the effect of kiss on GABA transmission to
GnRH neurons is due to pre or postsynaptic mechanisms. We performed whole-cell voltage-clamp recordings of GABA miniature
postsynaptic currents (mPSC) in GnRH neurons in the presence of
tetrodotoxin (TTX), which minimizes presynaptic actions by blocking action potentials. Kiss had no effect on the frequency of GABA
mPSCs, but did significantly increase the amplitude of these events.
To see if the effect of kiss on GABA PSC amplitude is mediated
postsynaptically, we recorded currents generated by local GABA application. Kiss significantly increased GABA-induced current amplitude. These data indicate that the kiss effect on frequency of GABA
transmission to GnRH neurons is due to presynaptic actions, but effects on amplitude are postsynaptic. Together with previous findings,
these data provide additional insights into the mechanisms of direct
and transsynaptic effects of kiss on GnRH neuronal activity.
TII.35
MATRIX METALOPRTEINASE 9 (MMP-9) PLAYS
A ROLE IN LONG TERM POTENTIATION (LTP) IN
AMYGDALAR BLA TO CE PATHWAY
Gorkiewicz T., Wawrzyniak M., Balcerzyk M., Kaczmarek L.
Matrix metalloproteinase-9 (MMP-9) is an extracelularly operating protein, capable to cleave several components of extracellular
matrix (ECM). It is expressed in neurons in many brain structures.
It has been shown to be important for maintenance of LTP in hipoocampal CA3 to CA1 pathway (Nagy et al. 2006) as well as in
the prefrontal cortex (Okulski et al. 2007). Amygdaloid body is a
small heteromeric structure, important for regulation of memory
and autonomic as well as endocrine responses. In the present study,
we have examined if LTP is MMP-9-dependent in the pathway
from basolateral to central amygdala. The basolateral nucleus of
amygdala (BLA) was theta burst-stimulated using a bipolar electrode, and EPSPs were collected from CE. We have found that in
slices from MMP-9 knock out mice the late phase of LTP is abolished. The same effect was obtained when inhibitor of MMP-9
was used in rat amygdala slices where LTP lasted for only 30 min
after its induction. Finally, we have checked LTP in slices from
the transgenic rats with neuron-specific MMP-9 overexpression,
driven by Synapsin I promoter. LTP in these rats was lower than in
control but stable. The present observation suggests that the proper level of MMP-9 expresion and activity is essential for synaptic
plasticity in the BLA-CE pathway, whereas MMP-9 overexpresion
may cause destabilization of neuronal environment and decreased
activity-dependent strengthening of synaptic transmission.
TII.36
SYNAPTIC CONNECTIONS BETWEEN PYRAMIDAL
CELLS AND INTERNEURONS IN SUPERFICIAL
LAYERS OF THE NEOCORTEX
Wozny C., Williams S.
Division of Neurobiology, MRC Laboratory of Molecular Biology,
Cambridge, UK
To understand how sensory information is processed in the neocortex
it is essential to investigate the neuronal microcircuits and the dynamics of cortical signalling. Layer 1 of the neocortex is a cell-sparse and
synaptic dense zone. Layer 1 neurons are exclusively GABAergic.
Here, we recorded from layer 1 interneurons in the adult neocortex
and we found that layer 1 interneurons displayed highly diverse morphological and electrophysiological properties. Electrophysiologically, L1 interneurons showed accommodating, non-accommodating,
accelerating, bursting, irregular and stuttering behaviour upon a sustained current injection. Based on their morphology, various types including neurogliaform cells, basket cells and axo-axonic cells could be
identified. Layer 1 interneurons are electrically coupled and provide
inhibitory synaptic input to fellow layer 1 interneurons and layer 2/3
pyramidal neurons. However, little is known about the translaminar
excitatory innervation of layer 1 interneurons. We performed pairedrecordings from layer 1 and layer 2/3 neurons in adult neocortical
slices. The properties of unitary EPSPs were found to be dependent
on the identity of the postsynaptic neuron. Our results suggest that
L2/3 pyramidal neurons efficiently recruit layer 1 interneurons providing a substrate for feedback inhibition, which might contribute to
the sparse firing of L2/3 pyramidal neurons observed in-vivo.
TII.37
EFFECT OF MATRIX METALLOPROTEASE
INHIBITION ON SHORT AND LONG TERM
PLASTICITY OF MOSSY FIBER-CA3 SYNAPSES
Wojtowicz T.1, Piotrowska A.2, Podhorska-Okolow M.2,
Dziegiel P.2, Kaczmarek L.3, Mozrzymas J.1
1
Laboratory of Neuroscience, Department of Biophysics,
2
Department of Histology and Embryology, Wroclaw Medical
University, Wrocław, Poland; 3 Department of Molecular and Cellular
Neurobiology, Nencki Institute of Experimental Biology PAS,
Warszawa, Poland
Matrix metalloproteinases (MMPs) are a large family of extracellularly-acting endopeptidases. MMPs were shown to play an
important role in hippocampal plasticity phenomena such as
LTP, learning and memory. In particular, in Shaffer collateral/
CA1 (Sch/CA1) synapses, MMPs are crucial for maintenance
of long term plasticity requiring NMDA receptor (NMDAR)
activation. However, it is not known whether MMPs affect
plastic changes in mossy-fiber/CA3 (mf/CA3) synapses, where
LTP is basically presynaptic and NMDAR-independent. Field
potential (fEPSPs) recordings in CA3 region (mf stimulation)
revealed no effect of FN439 (panmetalloprotease inhibitor) on
the short-term plasticity but the late phase of LTP was abolished
by this agent similar to Sch/CA1 pathway. Since maintenance of
LTP in Sch/CA1 was specifically dependent on MMP-9 activity, we have performed immunostaining for MMP-9 in mf/CA3
pathway. Semi-quantitative analysis revealed that induction of
LTP resulted in several fold increase in MMP-9 immunoreactivity within hilar and CA3 region relative to control slices. We
conclude that MMPs are crucial for long- but not short-term
plasticity in mf/CA3 synapses and that LTP induction in this
pathway is accompanied by a robust increase in MMP-9 immunoreactivity. It seems thus that consolidation phase of LTP
is critically dependent on MMP activity in the two major hippocampal pathways (Sch/CA1 and mf/CA3) where LTP induction mechanisms show profound differences. Support: Grant
No. P-N/030/2006.
TII.38
LACK OF PRODOMAIN ABOLISHES BDNF
CONSTITUTIVE SECRETION BY HEK 293 CELLS
Ziemlinska E.1, Skup M.1, Czarkowska-Bauch J.1, Bähr M.2,
Kügler S.2
1
Biology PAS, Warszawa, Poland; 2 Department of Neurology,
University Hospital Göttingen, Göttingen, Germany
Brain-derived neurotrophic factor (BDNF) and its proBDNF precursor are released both in constitutive and activity-dependent
manner. Prodomain itself proved to be necessary for BDNF targeting to regulated secretory pathway [Egan et al. (2003) Cell,
Chen et al. (2005) J Neurosci] but its role in constitutive secretion
is elusive. As mature BDNF (mBDNF) conveys trophic and prosurvival signals whereas proBDNF may convey growth inhibiting
and death signals, an important issue arises: can we control the
type of signal being triggered by BDNF? To verify this we cut off
the prodomain and generated plasmid coding only for rat mBDNF. To test the constitutive mBDNF construct secretion we have
chosen HEK 293 cell line. Two other plasmids coding either for
proBDNF (template for both BDNF forms) or proBDNF protected
from prodomain cleavage (source of proBDNF only), served as
controls. BDNF secretion was evaluated with WB technique using
antibodies detecting (1) both BDNF forms, (2) HA tag (mBDNF
construct) and (3) MYC tag (proBDNF constructs). We found all
three constructs being stably expressed in HEK cells. However,
in contrast to both proBDNF constructs, which were secreted and
detected in media fraction, mBDNF construct was revealed only
in the cell lysate fraction, not being released to the media. This is
the fi rst observation showing that mBDNF can be constitutively
released only when accompanied by prodomain. Support: ASTF
211-00-2007 for EZ, Polish-German grant to MS and SK.
TIII: Disorders of the Nervous System
TIII.01
INFLUENCE OF AGE UPON THE PHOTOTHROMBOTIC
STROKE IN MICE
Liguz-Lecznar M., Aleksy M., Skangiel-Kramska J.,
Kossut M.
Department of Cellular and Molecular Neurobiology, Nencki
Institute of Experimental Biology PAS, Warszawa, Poland
Stroke is one of the leading causes of death and disability in humans. Animal models of stroke simulate the human pathology
and can help to develop the effective therapies. One of such models is the photothrombosis model in which cerebral ischemia is
induced photochemically by injecting the photosensitive dye. The
advantages of this model are the small infarct size, minimal surgical manipulation of the animal. Despite the strong connection
of stroke with aging process, the majority of experimental studies are conducted in young or adult animals. Here we have used
young (3 months old) and aged (12 months old) mice to produce
the small (about 2 mm in diameter) infarct neighboring the primary somatosensory barrel cortex area and including the whole
depth of cortex. After 7-day reconvalescence we have analyzed
the presence and size of the evoked infarct. In both groups stroke
has evoked similar changes in cortex cytoarchitecture, however
we found that aging has decreased the method effectiveness of
about 25%. Moreover, comparing to young animals, in old mice
size and extent of infarct evoked with the same parameters and
conditions were much less unrepeatable. It can be associated with
changes of the nervous system physiology with age that concern
the blood flow, diameter of blood vessels, metabolism and physiology of neurons and glial cells. Alternatively, older mice may
have adapted functionally to age-dependent damage. Supported
by the Scientific Network: Biovision SNB-504-8.
TIII.02
ACTIVITY OF METALLOPROTEINASES IN THE
BRAIN OF YOUNG AND OLD MICE AFTER FOCAL
PHOTOTROMBOTIC STROKE
Aleksy M., Skangiel-Kramska J., Kossut M.
Department of Molecular and Cellular Biology, Nencki Institute
Aging is a major risk factor for ischemia. Old experimental animals demonstrate bigger infarct progression and more exten-
308
sive neuronal degeneration after stroke compared to young animals. Activation and expression of metalloproteinases (MMPs),
MMP- 2 and MMP-9 particularly, is strongly up-regulated after
stroke and it was proved to have a detrimental role. The present study was designed to compare MMPs activity in old and
young brains after ischemia. Cortical photothrombotic stroke
was induced in young (3–4 months) and aged (12–13 months)
C57/BL mice. MMPs activity on the infarcted area and surrounding tissue was examined at 1, 7 and 14 days after stroke.
Although in situ zymography revealed that the time-course of
gelatinolytic activity and the pattern of distribution of digested
products were similar in both groups, old mice exhibited stronger fluorescein signals of MMPs activity in the ischemic and
peri-infarct areas, as compared to young animals. Analysis of
brain homogenates by SDS-Page zymography showed the upregulation of MMP-9 activity after stroke in old brains, while
in young brains activity of this metalloproteinase was detected
at low level. MMP-2 activity remained at stable and comparable level in both groups at all investigated time-points. These
results suggest that old brains exhibit stronger proteolytic
reaction after stroke, which may contribute to a more severe
course of the disease. Supported by Polish-German Grant
P-N/026/2006 and Statutory Funds.
TIII.03
DISPARITY OF SEASONAL VOLUME CHANGES OF
FOREBRAIN STRUCTURES IN THE COMMON SHREW
(SOREX ARANEUS) SUGGEST ENGAGEMENT OF
DIFFERENT MECHANISMS.
Michalski A.1, Bartkowska K.1, Taylor J.2, Turlejski K.1
1
2
Department of Animal Ecology, Institute of Biology, University of
Bialystok, Bialystok, Poland
Mechanisms of seasonal changes of brain size in Sorex shrews
(Dehnel’s effect) remain largely unknown. Hypothesis postulating changes of brain cell numbers has been recently rejected
(Bartkowska et al. 2008). Other possible mechanisms are reorganisation of the neuropil and change in the composition of
brain tissue (e.g. dehydration). Shrews were captured in the
spring, summer and autumn. Some shrews collected in the
autumn wintered in captivity. Volumes of selected forebrain
structures were measured stereologically on Nissl stained series of sections. Largest oscillations were found in the volume
of cortex and basal ganglia. Lack of correlation between the
rate of neurogenesis and the volume of hippocampus confi rms
previous fi ndings. Relative quantity of myelin was determined
by densitometry of sections stained with the method of Gallyas.
Volumes of neocortex and hippocampus that are myelinised to
a similar degree change differently, while volumes of striatum
and neocortex, differing in myelin density, reduce their volumes
similarly in the winter. Moreover, the overall ratio of forebrain
volume to myelin quantity did not change throughout life. This
implies that general dehydration may be excluded as a significant reason of brain size alterations and that different mechanisms are engaged in various brain structures. The comparison
of analysed structures in terms of connectivity and water permeability by measuring levels of synaptophysin and aquaporin
4 was attempted.
TIII.04
PHONOLOGICAL AND SEMANTIC PROCESSING
IN CHILDREN WITH DEVELOPMENTAL DYSLEXIA
– AN EVENT-RELATED POTENTIAL STUDY
Jednorog K., Marchewka A., Tacikowski P., Grabowska A.
Event-related potentials were used to examine if the brain response (N400 component) in dyslexic children is modulated
by phonological or semantic priming, similarly to age-matched
controls. ERPs were recorded while the children listened to word
lists in which the semantic and phonological congruency of the
terminal words were manipulated. Dyslexics exhibited a dissociation of priming effects depending on whether semantically
or phonologically loaded primes were used. An enhancement
of N400 amplitude to semantically incongruent words was observed, although this effect was reduced and delayed compared
to that seen in controls. Direct comparison of the performance
of the two groups in the semantic priming task revealed that
they differed only in their response to semantically incongruent
words. In the phonological priming task, rather than an enhancement of the N400 amplitude found in controls, dyslexics displayed a reduction of the N400 to the incongruent condition in
comparison to the congruent condition. In this task, the studied
groups differed in both the phonologically congruent and incongruent conditions. These results suggest that when faced with
phonological priming, dyslexics have problems with both matching for similarities (integration into context) and incongruency
detection. In the case of semantic priming, the integration of
semantic context seems relatively intact in dyslexics, but they
experience difficulties in detecting the shift from one semantic
category to another.
TIII.05
HOW NEURONS BECOME GIANT – IDENTIFICATION
OF PROTEINS REGULATED BY mTOR KINASE IN
ETIOPATHOLOGY OF TUBEROUS SCLEROSIS
Malik A.1, Swiech L.1, Perycz M.1, Urbanska M.1, Blazejczyk M.1,
Wulf P.2, Parobczak K.1, Pietruszka P.1, Zarebska M.1,
Rutowicz K.1, Jaworski J.1
1
Laboratoty of Molecular and Cellular Neurobiology, International
Institute of Molecular and Cell Biology, Warszawa, Poland;
2
Departament of Neuroscience, Erasmus MC, Rotterdam,
The Netherlands
mTOR is a serine-threonine protein kinase for several aspects of
neuronal function. Increased mTOR signaling has been implicated in tuberous sclerosis (TS), a multiorgan disease affecting
brain. TS is caused by mutations in genes encoding for hamartin
and tuberin that lead to increase of the mTOR activity and subsequent abnormal cell growth and proliferation, leading to brain
lesions containing giant cells. It is unclear how mTOR contributes to observed changes. Our aim was to identify downstream
mTOR effectors important for the disease related abnormal cell
growth. To model TS giant cells, rat cortical neurons cultured
in vitro were transfected with short interfering RNA (siRNA)
targeting tuberin that caused a Rapamycin-sensitive increase of
neuron soma size. Next, we designed a siRNA library directed
against 140 mRNAs encoding potential mTOR targets, selected
based on published data. To select siRNAs decreasing soma
size of enlarged cells, cortical neurons were contransfected
with tuberin siRNA together with siRNAs from the library. Our
screen revealed over 20 genes, whose expression downregulation reversed the giant-cell like phenotype and 6 siRNAs that
further increased size of cells with tuberin knockdown. Among
proteins contributing to abnormal neuron growth upon mTOR
overactivation, we identified those involved in actin cytoskeleton dynamics, vesicular transport and cellular signaling. This
work has been financed by PBZ-MNiI-2/1/2005 and PNRF-96AI-1/07 grants.
TIII.06
VACCINE PRESERVATIVE, THIMEROSAL, AFFECTS
PAIN SENSITIVITY, BODY TEMPERATURE
AND EXTRACELLULAR GLUTAMATE IN THE
PREFRONTAL CORTEX IN RATS
Duszczyk M., Olczak M., Mierzejewski P., Majewska M.
Department of Pharmacology and Physiology of the Nervous System,
Institute of Psychiatry and Neurology, Warszawa, Poland
Mercury compounds are known neurotoxins. Thimerosal
(THIM), which contains molecularly 49% of mercury, is added
as preservative to many child vaccines and is suspected to be a
major factor in autism pathogenesis. Data from Adverse Event
Reporting of the Center for Disease Control and Prevention
(USA) provide epidemiological evidence for a link between
mercury exposure from THIM-containing vaccines and autism or other neurodevelopmental disorders. Our earlier studies showed that neonatally administered THIM causes persistent changes in pain reactivity, which are manifested in adult
rats. In this study we investigated acute effects of THIM on pain
reactivity, body temperature and brain glutamatergic system
in mature rats. Single injection of THIM induced marked hypoalgesia, measured in hot plate test. This effect was time- and
dose-dependent. It was reversed by administration of naloxone
before the test and by two neurosteroids – dehydroepiandrosterone sulfate and androsterone. THIM also caused a dose-dependent hypothermia. Administration of THIM directly to the
prefrontal cortex of freely moving rats increased the extracellular concentration of glutamate. The results suggest that acute
THIM injection rapidly changes the neurochemical systems related to glutamate, opioid peptides, and probably GABA, which
may contribute to developmental and neurotoxic effect of this
compound. Funded by EC grant MEXC-CT-2006-42371 to M.
D. Majewska.
TIII.07
EARLY POSTNATAL THIMEROSAL
ADMINISTRATION, IN A VACCINATION-LIKE
SCHEME, CAUSES VAST STRUCTURAL DAMAGE
IN RAT BRAIN: IMPLICATIONS FOR AUTISM
Olczak M., Duszczyk M., Mierzejewski P., Majewska M.
Department of Pharmacology and Physiology of the Nervous System,
Institute of Psychiatry and Neurology, Warszawa, Poland
Thimerosal (THIM), an organomercury compound added to
many child vaccines, is a prime suspect as agent causing au-
tism epidemic. Data analysis from Vaccine Adverse Event Reporting System (CDC,USA) revealed that children immunized
with THIM containing vaccines are several times more likely
to develop autism and other neurodevelopmental disorders than
those, who received THIM-free vaccines. In this study we examined developmental neurotoxic effects of THIM administered to
Lewis and Wistar rats i.m. in four equal doses (12 μg Hg/kg to
3 mg Hg/kg) on postnatal days 7–14. Analysis of Hg content
in brains of THIM-treated animals showed significant amounts
of Hg, which remained there for longer than 30 days. When
animals reached maturity their brains were removed and examined for histopathological changes using H&E and immunohistochemistry staining (GFAP, synaptophysin, neurofilaments,
dopamine, opiate receptors). Vast structural damage was found
in the brains of THIM-treated animals: reduced number of Purkinie cells, ischemic and necrotic changes in the amygdala,
ischemic and cell structure abnormalities in the temporal neocortex, dorsal and ventral hippocampus; hippocampal, pontal
and cerebellar clasmatodendrosis, loss of synaptic junctions
in hippocampus. These neuropathological changes correspond
with behavioral alterations observed in THIM-treated rats and
seem analogous to structural brain abnormalities found in autistic patients. Funded by EC grant MEXC-CT-2006-42371 to
M.D. Majewska.
TIII.08
TRANSIENT BRAIN ISCHEMIA AFFECTS BLOOD
LEVEL OF BRAIN-DERIVED NEUROTROPHIC FACTOR
IN EARLY REPERFUSION
Kravcukova P., Danielisova V., Nemethova M., Burda J.,
Cizkova D., Gottlieb M.
Institute of Neurobiology, Slovak Academy of Sciences, Kosice,
Slovak Republik
Brain-derived neurotrophic factor (BDNF) acts on certain neurons of the central and peripheral nervous system, helps to support the survival, encourage the growth and differentiation of
new neurons and synapses. In our study we monitored BDNF
blood level in course of brain ischemia-reperfusion in rat models
of transient focal (FI) and global (GI) ischemia. Blood samples
were collected before and during ischemia and at 40 and 90 min
of reperfusion. Results showed that in model of FI BDNF concentration in total blood, in plasma and blood cells rapidly decreased during fi rst 40 min of ischemia. In samples of whole
blood and blood cells BDNF started to rise at the end of ischemic
insult to control level, but in plasma it significantly decreased.
In model of GI we didn’t observe important changes after insult. During fi rst 90 min of reperfusion in both models BDNF
level in total blood and in blood cells continuously decreased.
Plasma level of BDNF started to rise at 40 min of reperfusion.
At 90 min of recirculation BDNF level in model of FI reached
92%, in GI about 155%. In conclusion we can state that brain
ischemia causes reduction of BDNF level during ischemia and
subsequent elevation of BDNF concentration in blood cells at
the end of ischemic insult followed by decreasing in early period
of reperfusion. On the other side, plasma level of this protein
reduced during ischemia markedly rises during recirculation.
Supported by VEGA 2/0141/09, VEGA 2/0146/09, APVV LPP023506, APVV-51-002105.
310
TIII.09
THE NEUROPROTECTIVE EFFECTS OF UROCORTIN
2 IN BILATERAL COMMON CAROTID OCCLUSION
INDUCED RETINAL DEGENERATION
Szabadfi K.1, Atlasz T.2, Reglodi D.3, Kiss P.3, Szabo K.1,
Danyadi B. 3, Tamas A.3, Fekete E.4, Zorrilla E.5, Gabriel R.1
1
Department of Experimental Zoology and Neurobiology,
2
Department of Sportbiology, 3 Department of Anatomy,
4
Department of Physiology, University of Pecs, Pecs, Hungary;
5
Department of Physiology, The Scripps Research, California,
Florida, USA
Urocortin 2 (Ucn 2) is a CRF paralog that preferentially activates
CRF2 receptors. Little is known regarding potential retinoprotective effects of Ucns despite the known presence of CRF family
peptides and their receptors in retina. We investigated the effects
of intravitreal Ucn 2 administration on ischemia-induced retinal degeneration (BCCAO). Two-month-old rats were subjected
to BCCAO and their retinas were processed histologically after
two weeks survival to determine the density of viable cells in the
ganglionic cell layer and the thickness of all retinal layers. Immunohistological analysis of PKC-, calretinin-immunoreactivity was
also performed. In BCCAO reduced retina thickness by approximately 60% as compared to sham-operated animals. Intraocular
Ucn 2 treatment led to a nearly intact appearance of the retinal layers, and the thickness of all layers was significantly increased by
40% compared to ischemic vehicle-treated subjects. Ucn 2 treatment also increased the number of cells by 55% in the ganglionic
cell layer as compared to those from carotid-occluded retinas of
vehicle-treated subjects. Ucn 2 also reversed the alterations found
in the pattern of immunocytochemical markers, such as calretinin
and PKC. These findings suggest that intraocular Ucn 2 treatment
may protect against ischemia-induced retinal degeneration, results
with potential therapeutic implications for ophthalmic diseases.
Support: OTKA: K72592; F67830; 78480; T061766; Richter.
TIII.10
LOSS AND RESTORATION OF PERINEURONAL NETS
AFTER CORTICAL PHOTOTHROMBOTIC STROKE
IN RATS
Karetko M., Nowicka D., Skangiel-Kramska J.
The distribution and density of cells enwrapped with perineuronal
nets (PNs) were examined in the neocortex after unilateral photothrombotic stroke. After different survival times PNs was visualized using Wisteria floribunda aglutinin (WFA) staining in the
infarct core, peri-infarct and remote cortical areas. Sham operated
rats and contralateral hemisphere to the stroke site from experimental rats served as controls. In the infarct core, 4 h after stroke
only few cells enwrapped with PNs could be detected and none of
numerous dying cells present showed PNs, as revealed by double
staining (WFA and FJ-C). No cells with PNs were found 24 h after
stroke. In the peri-infarct area, dramatic loss of PNs-enwrapped
cells occurred (96%) as compared with the contralateral site, and
such cells were almost absent after 24 h. However, 30 days after
stroke, a significant number of cells with PNs reappeared. In remote cortical areas, 4h after stroke a reduction in the number of
cells with PNs was observed. After 24 h the loss of PNs-enwrapped
cells was profound (63%) in comparison to intact hemisphere. However, 7d after stroke complete restoration of the number of cells
enwrapped with PNs took place. The density of cells with PNs was
similar to that found in the contralateral site and in sham operated
rats. These results showed the wide spreading and transient effect
of photothrombosis on PNs in remote cortical areas. Supported by
MNiSW Grant N3030300832/0474 and statutory funds.
TIII.11
RELATIONSHIP BETWEEN ACTIVITY OF MATRIX
METALLOPROTEINASE -2 AND -9 (MMP-2, MMP-9)
AND ISCHEMIA INDUCED NEUROGENESIS IN ADULT
GERBIL HIPPOCAMPUS
Wojcik L., Zalewska T.
In physiological conditions proteolytic remodeling of extracellular
matrix (ECM) participates in the stem cells development. Signal
derived from ECM may activate specific intracellular signaling
pathways which involve focal adhesion kinase (FAK), a key component responsible for the flow of information to the cell. In our study
we checked if there is a spatial and temporal relationship between
activity of two matrix metalloproteinases (MMP-2 and MMP-9)
and neural stem-cell development in gerbil hippocampus 1, 2 and 4
weeks after short-term global ischemia. Furthermore we also investigate the activation of FAK. Our results show that after the ischemia
there is a significant increase in the number of proliferating cells in
DG which correlates with the activation of MMPs. Whereas in the
damaged CA1 pyramidal layer MMP-2 and -9 activity decreases as
a result of delayed death of neurons. In the same region we have also
noticed a few immature neurons (NF-200 positive) 2 and 4 weeks
after reperfusion, however they did not attain maturity. On the other
hand we have noticed higher MMPs activity in the astrocytes surrounding the CA1 region. The diverse localization of MMP-2 and -9
activity in both cell types may be due to their different functions –
firstly with the proliferation of newborn cells and secondly with the
creation of the environment while the formation of post injury glial
scar. Supported by MSRHE grants: 1266/P01/2006/31
TIII.12
THE HMGB1 RECEPTOR RAGE MEDIATES ISCHEMIC
BRAIN DAMAGE
Muhammad S.1, Barakat W.1, Stoyanov S.2, Sajjad M.1,
Yang H.3, Tracey K.3, Bendszus M.4, Rossetti G.5, Nawroth P.P.2,
Bierhaus A.2, Schwaninger M.1
1
Pharmacological Institute, 2 Department of Internal Medicine,
University of Heidelberg, Heidelberg, Germany; 3 Feinstein Institute
for Medical Research, Feinstein Institute for Medical Research,
Manhasset, USA; 4 Department of Neuroradiology, University of
Heidelberg, Heidelberg, Germany; 5 HMGBiotech, Milan, Italy
In ischemic stroke, the necrotic core is surrounded by a zone of
inflammation, in which delayed cell death aggravates the initial
insult. Here, we provide evidence that the receptor for advanced
glycation end products (RAGE) functions as a sensor of necrotic
cell death and contributes to inflammation and ischemic brain
damage. The RAGE ligand high mobility group box 1 (HMGB1)
was elevated in serum of stroke patients and was released from
ischemic brain tissue in a mouse model of cerebral ischemia.
A neutralizing anti-HMGB1 antibody and HMGB1 box A, an
antagonist of HMGB1 at the receptor RAGE, ameliorated ischemic brain damage. Interestingly, genetic RAGE deficiency and
the decoy receptor soluble RAGE (sRAGE) reduced the infarct
size. In vitro, expression of RAGE in (micro)glial cells mediated
the toxic effect of HMGB1. Addition of macrophages to neural
cultures further enhanced the toxic effect of HMGB1. To test
whether immigrant macrophages in the ischemic brain mediate
the RAGE effect, we generated chimeric mice by transplanting
RAGE -/- bone marrow to wild-type mice. RAGE deficiency in
bone marrow-derived cells significantly reduced the infarct size.
Thus, HMGB1-RAGE signaling links necrosis with macrophage
activation and may provide a target for anti-inflammatory therapy in stroke.
TIII.13
NEUROPROTECTION BY AMINOGUANIDINE IN
HIPPOCAMPAL CA1 REGION AFTER MIDDLE
CEREBRAL ARTERY OCCLUSION (MCAO)
Danielisova V.1, Kravcukova P.1, Burda J.1, Domorakova I.2,
Nemethova M.1, Gottlieb M.1
1
Department of Neurochemistry, Institute of Neurobiology,
CE SAS, Kosice, Slovak Republic; 2 Department of Histology, Faculty
of Medicine, University of P.J. Safarik, Kosice, Slovak Republic
The present study examined the effects of a selective inducible
nitric oxide synthase inhibitor, aminoguanidine (AG) on neuronal
cell survival after middle cerebral artery occlusion (MCAO) in
hippocampal CA1 region. Transient focal cerebral ischemia was
induced in rats by 60 min MCAO, followed by 7 days of reperfusion. Saline as vehicle or AG at the dosage of 150 mg/kg i.p. was
administered immediately after occlusion and thereafter twice a
day for three days. The evaluation of infarcted volume was made
by 2,3,5-triphenyltetrazolium chloride (TTC). For comparison of
cellular viability we used Fluoro-Jade B and NeuN staining to examine the evolving phases of infarction induced by MCAO. Treatment with AG significantly reduced total infarct volumes by 55%
in comparison with saline group. AG significantly improved the
neurological outcome. The number of degenerating neurons was
markedly reduced in hippocampal CA1 region compared to groups
without AG treatment. These changes were seen in the ipsilateral
and contralateral hippocampus. In conclusion, the focal ischemia
affects the hippocampus, which responds bilaterally to the injury.
Our findings show that AG decreases ischemic brain damage and
improves neurological recovery after transient ischemia induced
by MCAO. It is suggested that treatment with AG may present
a potential therapeutic strategy for the treatment of traumatic
brain injury. This study was supported by VEGA 2/0141/09, VEGA
2/0146/09, VEGA 1/4237/07 and APVV LPP 0235-06.
TIII.14
IL-1β STIMULATES CYTOKINE SECRETION
BY PERIPHERAL LEUKOCYTES FOLLOWING
SUBARACHNOID HEMORRHAGE IN RATS
Jedrzejowska-Szypulka H., Woszczycka-Korczynska I.,
Olakowska E., Larysz-Brysz M., Lewin-Kowalik J.
Department of Physiology, Medical University of Silesia, Katowice,
Poland
Much evidence indicates that pathologies observed after subarachnoid hemorrhage (SAH), including vasospasm and neurological defects, are the effects of immuno-inflammatory processes
accompanying the hemorrhage. Following SAH, CSF concentration of cytokines increase, what may change the cerebral perfusion and destroy brain–blood barrier. These cytokines are
produced by activated leukocytes as well as hypoxic cells in the
brain. The aim of this study was to examine the role of IL-1β in
stimulation of peripheral leukocytes to increase secretion of cytokines after SAH. SAH was produced by injection of 150 μL of
autologous arterial blood into cisterna magna. In 50% of animals,
IL-1β activity was inhibited by intracerebroventricular administration of anti-rat IL-1β antibodies (SAH’ groups). Control group
consisted of sham-operated rats. Ninety minutes or 24 h following surgery, blood samples were collected and subpopulations
of mononuclear leukocytes were isolated and cultured for 24 h.
Then, the concentration of TNF-α, ET-1 and IL-6 in supernatants
was measured by means of ELISA. SAH resulted in significant
increase in TNF-α and ET-1, especially after 24 h, and no change
in IL-6 concentration. Inhibition of IL1-β decreased the secretion of TNF-α and ET-1 and led to increase in IL-6. We found
that peripheral leukocytes are an additional source of cytokines
following SAH, and their activation depends on IL1-β produced
after hemorrhage.
TIII.15
ALTERATION OF UBIQUITIN IMMUNOREACTIVITY
AFTER ISCHEMIC POSTCONDITIONING AND EGb 761
TREATMENT IN CA1 HIPPOCAMPAL EURONS IN RATS
Domoráková I.1, Mechírová E.1, Danielisová V.2, Kravčuková P.2,
Burda J.2
1
Department of Histology and Embryology, P.J.Safarik University,
Kosice, Slovak Republic; 2 Institute of Neurobiology SAS, Kosice,
Slovak Republic
Ischemic postconditioning used at the right time after initial ischemia is able to protect the most vulnerable CA1 hippocampal neurons. Alteration of ubiquitin immunoreactivity
(UIR) was studied after 7 days of reperfusion in 4 experimental
groups of rats (1) 10 min of ischemia, (2) 10 min of ischemia
followed by ischemic postconditioning (5 min of ischemia done
after 2 days of reperfusion). Extract of Ginkgo biloba (EGb
761, 40 mg/kg; p.o.) was administered 5 h after 10 min of ischemia both in ischemic group (3) or ischemia/postconditioning
group (4). Quantitative analysis showed significant decrease
(45.0 ± 9.7) of ubiquitin positive CA1 neurons in group (1) in
comparison to sham control (187.5 ± 15.1) and nonsignificant
increase of neuronal UIR (74.8 ± 22.4) in group (3) with EGb
761 administration. Ischemia/postconditioning (2) showed neuroprotection and significant increase of ubiquitin positive CA1
neurons (133.4 ± 14.3). Ischemia/ postconditionig combined
with antioxidant EGb 761 (4) revealed suppression of neuroprotective effect of delayed ischemic postconditioning. Moreover,
significant decrease of ubiquitin positive neurons (10.8 ± 2.4)
in CA1 region was observed. Our results suggest that delayed
ischemic postconditioning is able to prevent ischemia induced
neurodegeneration while antioxidant used after initial ischemia
followed by postconditioning fully abolishes this effect. This
study was supported by VEGA grants: 1/4237/07, 2/0141/09 and
APVV LPP 0235-06.
312
TIII.16
PRECONDITIONING AND POSTCONDITIONING
EXERT STROKE NEUROPROTECTION MODULATING
NCX AND ASIC1a EXPRESSION
Pignataro G., Esposito E., Cuomo O., Sirabella R., Di Renzo G.,
Annunziato L.
Department of Neuroscience, Federico II University of Naples,
Naples, Italy
Ischemic preconditioning is a neuroprotective mechanism in which
a brief non-injurious episode of ischemia protects the brain from a
subsequent lethal insult. Recently, it has been reported that modified
reperfusion subsequent to a prolonged ischemic episode may also
confer neuroprotection, a phenomenon termed postconditioning.
Mitogen Activated Kinases (MAPK) play a key role in these neuroprotective mechanisms. Objectives of this study was to evaluate the
effect of MAPK activated by preconditioning and postconditioning
on the expression of Na+/Ca2+ exchangers(NCXs), and Acid Sensing
Ionic Channels (ASICs), two families of proteins that, by modulating intracellular ionic homeostasis, contribute to the progression of
the ischemic lesion. NCX and ASIC protein expression was investigated in rats subjected to harmful ischemia, preconditioning or postconditioning, treated with specific MAPK inhibitors or with vehicle
alone. Results of this study indicated that NCX1, NCX3 and NCKX2
are up-regulated in those brain regions protected by preconditioning
and postconditioning treatment while ASIC1a is downregulated in
the same regions. The change observed in the expression of these
proteins seems to be due to p-AKT. In fact, p-AKT inhibition reverted the preconditioning and postconditioning neuroprotective effect
and prevented NCXs overexpression and ASIC1a downregulation.
TIII.17
SYSTEMIC TREATMENT WITH FRESHLY ISOLATED
HUCB-MNs CELLS IS THE MOST EFFECTIVE IN
RESTORING FUNCTION FOLLOWING BRAIN DAMAGE
IN RATS
Pawlak E., Janowski M., Habich A., Jablonska A., Lukomska B.,
Domanska-Janik K.
The aim of the study was to compare therapeutic effectiveness of
intra-arterial infusion of human umbilical cord-blood derived mononuclear (HUCB-MNs) cells at different stages of their neural conversion in vitro. Freshly isolated HUCB-MNs (D-0) neuraly directed
progenitors (D-3) obtained during 3 days culture of HUCB-MNs
and neural stem cells (NSC) line derived from HUCB-MNs were
assessed. Focal brain damage of dorsolateral striatum was induced
in Wistar rats by stereotactic injection of previously established low
dose of ouabain (1 μl or 1,5 μl 5 mmol). Three days later 107 HUCB
cells were infused into carotid artery. Following surgery rats were
housed in large enriched environment cages, in groups of 7–8 animals per cage, for 30 days. Behavioral assessing consisted of: tests
for sensorimotor deficits (walking beam task, rotarod), habit learning
task, exploratory behavior (open field test) and apomorphine induced
rotations. Functional effects of different subsets of HUCB cells were
diverse in various behavioral tests and hard to conclude which stage
of neural conversion of cord blood cells is the most effective in functional recovery. Additional analysis was applied: scores concerning
positive effects of cells treatment visible in all parameters were calcu-
lated. The sum of scores revealed that the most effective in functional
restoration and reduction of lesion volume were freshly isolated D-0
HUCB cells. Supported by Medical Research Centre statutory fund.
TIII.18
PROTEIN KINASE C BETA IN BRAIN MITOCHONDRIA
Kowalczyk J.1, Debski J.2, Zablocka B.1
1
Molecular Biology Unit, Mossakowski Medical Research Centre
PAS, Warszawa, Poland; 2 Mass Spectrometry Lab, Institute of
Biochemistry and Biophysics PAS, Warszawa, Poland
PKC is implicated in the regulation of mitochondrial membranes integrity, however its precise function is not fully understood. We have
examined association of PKCβ with mitochondria isolated from
ischemia-vulnerable (CA1) and ischemia-resistant (CA2-3, DG) hippocampal regions in gerbil model of transient brain ischemia. We
show that ischemia-induced translocation of PKCβ to mitochondria
in (CA2-3,DG) is significant, rapid and stable and, may bespeak neuroprotection. Furthermore, in an in vitro organotypic hippocampal
slice culture, the specific PKCβ inhibitor - peptide increases detrimental effect of 100 μM NMDA. Cell death measured with propidium iodide showed 96% ± 25 vs. 63% ± 9 after NMDA alone and
vs. 1.5% ± 0.5 dead neurons in unchallenged slices. We have also
been looking for PKCβ mtiochondrial partners using “pull-down”
method and Mass Spectrometry analysis. It revealed mitochondrial
proteins: creatine kinase, ANT, α, β chains of ATP synthase, complex I protein of electron transport chain and thioredoxin, which
can bind directly to PKCβ. All of these proteins are connected with
energy and superoxide production. Our data suggest that PKCβ
translocation to mitochondria might be connected with endogenous
neuroprotection in CA2-3, DG hippocampus. PKCβ acting through
abovementioned mitochondrial proteins could positively influence
the balance in ROS production during reoxygenation after transient
ischemia. Supported by Polish Mitochondrial Network MitoNet.pl.
TIII.19
THE INFLUENCE OF ISCHEMIA/REPERFUSION
ON THE NEURONAL NITRIC OXIDE SYNTHASE
IMMUNOREACTIVITY IN LUMBOSACRAL SPINAL
CORD AND DORSAL ROOT GANGLIA
Schreiberova A., Davidova A., Capkova L., Lukacova N.
Spinal Cord Injury, Institute of Neurobiology, Slovak Academy of
Sciences, Kosice, Slovak Republic
Nitric oxide, a diffusible molecule which is constitutively expressed by
neuronal isoform of nitric oxide synthase (nNOS) acts as an important
non-conventional neurotransmitter in the nervous system. The distribution of nNOS-immunoreactive (-IR) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) positive neurons was studied by
immunohistochemical and histochemical methods in upper lumbar (L1L3) and lower lumbar and sacral (L4-S4) dorsal root ganglia (DRGs)
and in relevant spinal cord segments after 15 min ischemia followed by
7 and 14 days of reperfusion. The intensity of nNOS-IR and NADPHd
positivity was evaluated by densitometric analysis, using a software
UTHSCSA Image Tool. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons
in the spinal cord to transient ischemia and the following reperfusion
periods. In addition, our results clearly show the increase of nNOS-IR/
NADPHd positivity in spinal ganglia laid outside the spinal cord. Distri-
bution character of nNOS-IR/NADPHd positive fibers coming out from
DRGs reflects the increase of nNOS-IR and NADPHd positivity in thin
and thick myelinized axons of dorsal roots and dorsal funiculi. These
results explain the participation of nitrergic system in nociceptive and
proprioceptive circuits in the spinal cord. Supported by the APVV 0314
-06 and VEGA grants 2/0110/08 and 2/0015/08.
TIII.20
RESTITUTION OF SENSORY FUNCTION OF
VIBRISSAE AFTER STROKE IN THE BARREL FIELD
COINCIDES WITH ACTIVITY CHANGES IN SPARED
SOMATOSENSORY CORTICAL AREAS
Jablonka J.1, Nosecka E.2, Kossut M.1
1
Department of Molecular and Cellular Neurobiology, 2 Department
of Neurophisiology, Nencki Instute of Experimental Biology PAS,
Warszawa, Poland
We described previously a sequence of cortical activity changes after
the unilateral stroke in the whiskers somatosensory representation –
the barrel field (BF). We aimed to check if this activity remodeling
is linked to the restitution of the whiskers sensory function. To characterize poststroke deficits and time related recovery we performed
the gap-crossing test for a group of rats 3, 28 and 56 days after the
photothrombotic stroke (DPS) in the BF. Rats learned to cross the
gap between elevated platforms in darkness, using only sensory information from whiskers. We examined the minimum distances at
which rats failed to cross (min) and the maximal distances (max)
crossed by the rats in two scenarios at each time point: (1) with all
their whiskers and (2) only with unilateral whiskers corresponding
to the destroyed BF (contralateral to the stroke). Before the stroke
there were no significant differences in min and in max distances
when rats used all versus unilateral whiskers. At 3DPS the max was
lower when rats used whiskers with destroyed sensory representation. The first fall was made by rats at shorter distances when they
used just the impaired side whiskers. At 56DPS all the differences
between crossing performance disappeared. At this time point, we
previously found new activity foci in the spared somatosensory cortex of the injured hemisphere, specific for the whiskers stimulation
– probably a new functional representation of the whiskers.
TIII.21
EXPERIMENTAL NEUROPHYSIOLOGICAL MOTOR
EVOKED POTENTIAL STUDIES IN THE ISCHEMIC
STROKE RAT’S MODEL
Szukala A.1, Huber J.1, Czarnecki P.2, Romanowski L.2
1
Department of Pathophysiology of Locomotor Organs, 2 Department
of Hand Surgery and Traumatology, Karol Marcinkowski University
of Medicine in Poznan, Poznań, Poland
This study was undertaken to ascertain functional changes in areas of
ipsi- and contralateral motor cortex involved with the ischemic incidences following experimental closing of flow in cervical aorta uni(right) or bilaterally when recordings of motor evoked potentials induced with magnetic field (MEPs) were performed.Aims of this study
were (1) ascertaining changes in parameters of recordings in motor
evoked potentials (MEPs) following experimental ischemic conditions, (2) histological verification of range in pathological changes
within motor cortex neurons following ischemia, (3) estimation the
correlation between duration of ischemia and changes in activity of
cortico-spinal neurons. Studies were performed on 24 Wistar rats. Recordings of MEPs induced in the left motor cortex were performed
from right sciatic nerve.Significant fluctuations in MEPs amplitudes
from 4.5 mV to 1.5 mV were commonly found within 8 minutes from
the experiment onset what might indicate on changes in excitability
and transmission of certain number corticospinal neurons. Contrary
to the previously found changes in MEPs latencies we observed more
often the reversible changes of amplitude parameter. The time of 6
minutes after the significant ischemic incidence is not critical for irreversible and persistent changes in activity of cortico-spinal neurons.
TIII.22
THE EFFECTS OF CPPG ON SPATIAL MEMORY AND
THE ACTIVITY OF MMP-2 AND MMP-9 IN
THE HIPPOCAMPUS OF RATS WITHOUT OR AFTER
HYPOXIA
Car H.1, Michaluk P.2, Wiśniewska R.1, Kaczmarek L.2
1
Department of Pharmacology, Medical University of Białystok,
Białystok, Poland; 2 Laboratory of Molecular Neurobiology,
CPPG is a highly potent group III metabotropic glutamate receptor
(mGluR4/6/7/8) antagonist with selective affinity to mGluR4. The aim
of the present study was to determine the effects of CPPG on spatial
memory in the water maze, and on the activity of MMP-2 and MMP9 metalloproteases in the hippocampus of rats without and after hypoxia. Short-term hypoxia (2% O2 and 98% N2) enhances excitatory
synaptic transmission and impairs spatial acquisition in the water
maze. We observed that CPPG prolonged only escape latency during the first day of training in water maze in group without hypoxia,
while it did not influence escape latency, distance travelled and
swim speed during training of rats after hypoxia. It did not change
previously acquired escape response during free-swim trial in either
group of rats. Hypoxia significantly increased the activity of the proform of MMP-9 in the hippocampus. Gel zymography showed elevations of MMP-2 and MMP-9 activity in the hippocampus of rats
without or after hypoxia when CPPG was administered. Concluding,
CPPG has no effects on deficits of behavioural processes induced by
hypoxia in the water maze. Enhanced activity of MMP-2 and MMP9 in the hippocampus induced by CPPG does not correspond to the
influence on acquisition in the water maze. The reconfiguration of
extracellular matrix by MMP-2 and MMP-9 in hippocampus is new
central effect of CPPG. Support: grant No 3-10614L from the State
Committee for Scientific Research, Warszawa, Poland.
TIII.23
IMMUNOSUPPRESSANTS ATTENUATE HMGB1
EXPRESSION AND RELEASE FROM PRIMARY
ASTROCYTE CULTURES EXPOSED TO COMBINED
OXYGEN-GLUCOSE DEPRIVATION
Gabryel B., Bielecka A., Bernacki J.
Department of Pharmacology, Medical University of Silesia,
Katowice, Poland
Protective potential of immunosuppressants has been reported in
many experimental models of ischemia both in vivo and in vitro
suggesting novel therapeutic application of these drugs. On account
of fact that high mobility group box 1 (HMGB1) protein has recently
314
been reported to be involved to ischemic brain injury, the purpose
of the present study was to determine whether treatment with immunosuppressants could decrease HMGB1 expression and release
in astrocytes exposed to ischemia-simulating conditions (combined
oxygen glucose deprivation, OGD). We also studied the influence of
these drugs on expression of NFκB, inducible NO synthase (iNOS)
and cyclooxygenase-2 (COX-2). In addition, we investigated whether the immunosuppressants could attenuate of necrosis in astrocyte
cultures exposed to OGD. Cells were treated with cyclosporine A,
FK506 and rapamycin (all drugs at concentrations of 0.1, 1 and 10
mM). Our study has provided evidence that immunosuppressants
decrease the expression and release of HMGB1 in ischemic astrocytes. The present results provide further information about the cytoprotective mechanisms of immunosuppressants towards ischemic
astrocytes, in relation to the pathophysiology of ischemic brain
injury. It appears that the immunosuppressants stimulated protective effects could be mediated in part by suppression of HMGB1
expression and release in astrocytes, what leads to attenuation of
ischemia-induced necrosis and neuroinflammation.
TIII.24
MOLECULAR MECHANISMS OF AXON
DEGENERATION AND ITS BLOCKADE
Beirowski B.
Department of Neurology, University of Goettingen, Goettingen,
Germany
Axon degeneration is a critical feature in peripheral neuropathies and
in neurodegenerative diseases. A valuable model for study of axon
degeneration is that of Wallerian degeneration (WD) in which the
distal portion of a transected axon undergoes a sequential process of
degeneration. The discovery of the WldS mouse mutant which exhibits strong delay in WD due to expression of nuclear WldS protein has
completely changed our understanding of WD which is now regarded
as an active auto-destruction programme. Dissection of the cellular
and molecular mechanisms of this novel death pathway is paramount
for the development of new therapies. I will summarise my recent
work which shed new light on the mechanisms of WD, chronic axon
pathology and the mode of action of WldS [e.g. Beirowski et al. (2009)
J Neurosci 29: 653–668, Beirowski et al. (2008) Eur J Neurosci 28:
1166–1179].
TIII.25
DYNAMICS OF CHANGES OF EXECUTIVE FUNCTIONS
IN PATIENTS WITH TRAUMATIC BRAIN INJURIES
Dzierzak E.
Faculty of Psychology, University of Warsaw, Warszawa, Poland
The purpose of the study was to observe the dynamics of changes of
executive functions in patients with light and mild traumatic frontal
lobes injuries. Patients, with changes indicated in CT, and the control
group accordingly, were presented with a set of neuropsychological
methods (i.e. Tower of London (TOL), Trail Making Test (TMT) ,
Stroop Test etc.), three times during three weeks following the brain
trauma and after half a year after the brain trauma. Dynamics of
change may differ according to individual factors (Leon-Carrion et
al.), however in results obtained an interesting pattern of improvement may be observed. Namely, during the short period after the
brain trauma, patients’ level of functioning reaches its peak in the
first and third week of recovery, whereas decreases rapidly during
the second week (e.g. see TOL, F1, 14=2.5, P<0.05). Above results may
be helpful in the future evaluation of methods in neuropsychological
rehabilitation.
TIII.26
THE EFFECT OF NOISE EXPOSURE IN RAT PUPS ON
THE ACOUSTIC STARTLE REFLEX IN ADULTHOOD
Rybalko N., Burianová J., Bureš Z., Syka J.
Department of Auditory Neuroscience, Institute of Experimental
Medicine, Prague, Czech Republic
Acoustical trauma during the sensitive period of postnatal development may result in the impairment of the auditory system,
manifested as deteriorated auditory sensitivity and abnormalities in the processing of auditory signals. The acoustic startle
reflex (ASR) and the prepulse inhibition (PPI) of the ASR were
examined in 3–6-month-old rats exposed to broad-band noise
(125 dB SPL, 8 min) in early postnatal life (day 14). Hearing
thresholds in noise-exposed rats, examined by measurements of
auditory evoked responses, were found to be normal. The ASR to
tones in the range of 2–16 kHz and the PPI of the ASR to a noise
burst (110 dB SPL) induced by 2, 4, 8, 12 and 16 kHz tones over
a range of 20–80 dB SPL were measured in exposed rats and in
age-matched controls. In all exposed rats, a decreased strength
of the startle reflex in response to intense stimuli was observed
in comparison with controls. The effect of acoustic trauma on the
PPI was different from the effect on the ASR: tones of 20–30 dB
SPL were able to suppress the ASR in exposed rats significantly
more than in control animals, a similar tendency was observed
at higher intensities. The results indicate that acoustic trauma
in the early developmental period induces abnormalities in the
auditory system of rats in the processing of stimulus intensity in
addition to abnormalities in the processing of sound frequency
[Grécová et al. (2009) Eur J Neurosci, in press]. Supported by
AV0Z50390512, GACR 309/07/1336, LC 554.
TIII.27
CONSERVED MECHANISMS OF GENE CISREGULATION FOLLOWING STROKE AND SEIZURES
Dabrowski M.1, Dojer N.2, Zawadzka M.1, Kaminska B.1
1
Laboratory of Transcription Regulation, Nencki Institute of
Experimental Biology PAS, Warszawa, Poland; 2 Institute of
Informatics, University of Warsaw, Warszawa, Poland
We performed SVD analysis of our data from expression profiling in the rat MCAO model of stroke and of published dataset
from the rat hipopocampus following kainate-induced seizures.
The contributions (across the genes) of two SVD modes were
highly conserved following stroke and seizures, suggesting that
they reflect mechanisms of gene regulation conserved following seizures and stroke. Functional annotation revealed that the
fi rst conserved SVD mode reflects inflammatory response and
apoptosis. The second conserved mode reflects increased expression of genes associated with neuronal synaptic activity. Applying Bayesian networks learning algorithm to the data from
Transcription Regulatory regions And Motifs (TRAM) database, we looked for putative cis-regulatory features associated
with the conserved modes. We report that two motifs, binding
transcription factor AP1, and the matrix-attachment region binding protein SATB1, had opposing effects on gene expression in
the subspace of the mode reflecting inflammation and apoptosis.
A number of features; binding transcription factors associated
with neuronal plasticity (Egr1-3, Creb, Zfp161), hypoxia/oxidative stress (Nfe2l1-2, Arnt), and neuronal differentiation (Lhx13); were associated with the mode reflecting neuronal activity.
The results obtained on one dataset were confi rmed on the other,
which validated the features and – more importantly – revealed
mechanisms of gene co-regulation conserved between stroke and
seizures.
TIII.28
GELATINOLYTIC ACTIVITY IS PRESENT IN THE
INTERCHROMATIN SPACE OF THE NEURONAL CELL
NUCLEUS
Gawlak M.1, Gorkiewicz T.2, Wilczynski G.1
1
Department of Neurophysiology, 2 Department of Molecular and
Cellular Neurobiology, Nencki Institute of Experimental Biology
Matrix metalloproteinases (MMPs) form an enzyme family
which, by mainstream research, is implicated in extracellular
matrix processing in physiological and pathophysiological conditions. Some of these proteins termed gelatinases, in particular
MMP-2 and MMP-9, cleave gelatin as an artificial substrate.
Surprisingly, a number of studies have revealed the presence
of gelatinolytic activity in the cell nucleus. Although the phenomenon appears not to be artifactual, neither the identity nor
the role of nuclear gelatinases has been established unequivocally. In the nervous system, nuclear gelatinolysis is detectable
in normal conditions, yet it is induced by seizures, and stroke.
We studied nuclear gelatinolytic activity by high resolution
in situ zymography (ISZ) in sections of alcohol-fi xed, polyester wax-embedded normal rat brain. Ubiquitously distributed
among the major brain areas the ISZ signal was present mainly
in neurons. At high magnification, our study revealed previously
unrecognized mesh-like pattern of nuclear gelatinolytic which,
by counterstaing with fluorescent DNA-binding dye, represents
an interchromatin space. The ISZ signal colocalized with the ribonucleoprotein compartment enriched in splicing components,
identified using an immunoreactivity of spliceosome assembly
factor SC-35. This suggesting a function for MMPs in processes
of gene-expression and/or RNA-processing and hypothetically
involvement in remodeling of chromosome territories.
TIII.29
THE NEUROPROTECTIVE EFFECTS OF PACAP IN
SEVERAL MODELS OF NEURODEGENERATION IN
THE RAT RETINA
Atlasz T.1, Szabadfi K.2, Reglodi D.3, Kiss P.3, Gaal V.4, Tamas A.3,
Toth G.5, Szabo K.2, Molnar A.2, Gabriel R.2
1
Department of Sportbiology, 2 Department of Experimental Zoology
and Neurobiology, 3 Department of Anatomy, 4 Department of
Ophtalmology, University of Pecs, Pecs, Hungary; 5 Department of
Medical Chemistry, University of Szeged, Szeged, Hungary
Pituitary adenylate cyclase activating polypeptide (PACAP)
is a neurotrophic and neuroprotective peptide that has been
shown to exert protective effects in different neuronal injuries,
models of neurodegenerative diseases and cerebral ischemia.
PACAP and its receptors are present in the retina. PACAP is
neuroprotective in several models of retinal degeneration in
vitro and in vivo. In the present study we summarize the protective effects found with intravitreal PACAP treatment in the
following models of retinal degeneration in rats: (1) toxic injury
induced by monosodium glutamate (MSG) treatment; (2) ischemic injury (BCCAO) and (3) degeneration induced by different kind of UV-A light. MSG leads to the severe degeneration
of the inner retinal layers. BCCAO cause degeneration in all
retinal layers. UV-A exposure leads to a severe damage of the
outer layers of the retina. In all cases, intravitreal PACAP injection (100 pmol) was administered into one (right) eye, while
the other eye (left) received saline treatment, serving as control
retinas. Histological and immunocytochemical analysis of the
retinas showed that PACAP treatment significantly ameliorated
the damaging effects of all treatments. These results provide
the basis for future clinical application of PACAP treatment in
retinal degeneration. Support: OTKA: K72592; F67830; 78480;
T061766; Gedeon Richter Ltd.
TIII.30
PROTECTION OF HIPPOCAMPAL NEURONS AGAINST
NEUROTOXIC DAMAGE BY ANTI-MYELIN T CELLS
MAY INVOLVE DIMINISHING THE ACTIVATION OF
MICROGLIA AND NG2+ GLIAL CELLS
Kurkowska-Jastrzebska I.1, Zaremba M.2, Oderfeld-Nowak B.2
1
2nd Department of Neurology, Institute of Psychiatry and
Neurology, Warszawa, Poland; 2 Department of Molecular and
We have previously demonstrated that CD4+ anti-myelin basic protein (MBP) T cells protect hippocampal neurons against
trimethyltin-induced damage. In this study, we employ immunocytochemicalmethods to investigate the influence of administration of T cells to the response of microglia and of NG2+
cells to TMT-induced damage. Female Lewis rats were treated
with anti-MBP CD4+ T cells (4 million per animal, i.v.) 24
hours after TMT (8 mg/kg, i.p.) intoxication. TMT caused degeneration of CA4 hipppocampal neurons and evoked an abundant reaction of microglial and NG2+ cells in the injured zone.
The number of activated cells increased about 4-fold relative
to controls as assessed on the 21st day after TMT treatment.
A bulk of the cells of ameboid morphology, which expressed
NG2 or microglial antigens, appeared in the zone of neurodegeneration. Interestingly, many of the cells of ameboid phenotype shared both antigens. Administration of T cells downregulated the activation of both glial classes and considerably
reduced the formation of the ameboid phenotype forms. The
latter were decreased, on average, by 60 percent. Our data
suggest that the diminished activation of microglia and NG2+
cells, particularly the decrease of the ameboid forms known to
release a number of proinflammatory substances, may contribute to the neuroprotective effect of T cells. Supported by the
grant no N401-1293-33 of the Ministry of Scientific Research
and Information Technology in Poland.
316
TIII.31
ANALYSIS OF PUTATIVE TOXIC INTERACTION OF
1-METHYLNICOTINAMIDE WITH DOPAMINERGIC
NEURONS
Slomka M., Ziembowicz A., Lazarewicz J.
It has been proposed that in the Parkinson’s disease exo- or endogenous N-methyl-compounds like 1-methylnicotinamide (MNA)
could be accumulated by dopaminergic neurons and induces neurotoxic effects, as happens with MPP+. The aim of this study was
to test this hypothesis by comparing direct effects of MPP+ or
MNA application into the striatum of 7 day old rat pups and to the
substantia nigra (SN) of the adult rats. Microinjections of tested
substances in doses: MPP+ (20 and 100 μg) and MNA (20, 40, 80
μg), were made unilaterally. Effects of MNA and MPP+ on pups’
striatum were evaluated by TTC staining and the lesion volume
was calculated using the ImageJ program. In the SN of adult rats
alterations in the level of tyrosine hydroxylase (TH) immunostaining were examined at the 5th day after MPP+ or MNA injection, utilizing antibody specific to TH. Moreover the coronal sections (20 μm thick) of the SN were examined to evaluate MNA- or
MPP+-induced glial activation with the glia-specific lectin. Using these methods we did not observe any toxic effect of MNA.
MPP+ induced statistically significant damages in the striatum of
rat pups, whereas there was no lesion after MNA injection. Also
in the SN of adult rats MPP+ caused 50% loss of TH-positive neurons and doubled the number of activated glial cells, while MNA
injection had no visible effect. Summing up, present data did not
demonstrate any toxic effects of MNA on dopaminergic neurons
in the rat brain in vivo.
TIII.32
THE ROLE OF LOW MOLECULAR WEIGHT THIOLS
IN THIMEROSAL TOXICITY – AN IN VITRO STUDY
Zieminska E.1, Toczylowska B.2, Wojcik J.2, Stafiej A.1, Bal W.3,
Lazarewicz J.1
1
Centre PAS, Warszawa, Poland; 2 Laboratory of Biological NMR,
3
Department of Biophysics, Institute of Biochemistry and Biophysics,
Warszawa, Poland
Thimerosal (TH), an ethylmercury complex of thiosalicylic acid
has been used as preservative in vaccines. Inspired by a known
high affinity of mercury for thiol groups, we examined whether
the presence of L-cysteine (Cys), D,L-homocysteine (Hcy), Nacetyl cysteine (NAC), L-methionine (Met) and glutathione (GSH)
in extracellular space could influence the viability, intracellular
calcium concentration ([Ca2+]i) and mitochondrial membrane potential in rat cerebellar granule cells. The cells were exposed to
500 nM TH for 48 h or 15 μM TH for 10 min. The loss of cells viability could be prevented partially or wholly, in a dose-dependent
manner, by 60, 120 or 600 μM Cys, Hcy, NAC and GSH, but not by
Met. The elevation in [Ca2+]i and mitochondrial potential induced
by 25 μM TH were abolished by all compounds studied, except
for Met, at 600 μM. The loss of the ethylmercury moiety from TH
as a result of interaction with thiols studied was monitored by 1Hand 199Hg-NMR spectroscopy. This extracelullar process may be
responsible for the neuroprotection seen in cerebellar cell culture,
but also provides a molecular pathway for redistribution of TH
derived toxic ethylmercury in the organism.
TIII.33
LENTIVIRAL DELIVERED siRNA AGAINST ICER
PROTECTS PRIMARY CORTICAL NEURONS FROM
APOPTOSIS CAUSED BY SERUM DEPRIVIATION
Klejman A., Bieganska K., Gierej D., Kaczmarek L.
of Experimantal Biology PAS, Warszawa, Poland
CREB activation and CREB-dependent signaling pathways are
crucial for neuronal survival. The term ICER (inducible cAMP
early repressor) refers to four protein isoforms that are all endogenous, inducible antagonists of CREB. It was previously shown,
that all 4 ICER isoforms are induced upon pro-apoptotic treatment,
and also that each of them separately evokes neuronal cell death in
cortical culture transfected with these genes. The ICER proteins
are believed to be strong repressors of Immediate Early Genes,
which are involved in cell response to inter- and/or intra-cellular
signals. Herein, we have applied the siRNA approach to silence
ICER expression. Because ICERs are members of CREM family
of proteins, sharing with them the gene sequence, only the small
unique region for ICER was selected to design ICER-directed,
specific siRNA. Indeed, we obtained functional siRNA capable of
blocking ICERs but not affecting CREM proteins. With this tool,
we have investigated if the ICER’s silencing protects neurons from
apoptosis caused by either serum deprivation or excitotoxicity. Using the lentiviral vector, as a vehicle to deliver siRNA (shRNA) we
have found that silencing of ICER mildly, although significantly,
protects primary cortical neurons from apoptosis caused by serum
deprivation.
TIII.34
IMPAIRMENT OF CHOLINERGIC SEPTAL SN56 CELLS
BY THIAMIN DEFICIENCY
Bizon-Zygmanska D., Bielarczyk H., Jankowska-Kulawy A.,
Szutowicz A.
Gdańsk, Poland
Cholinergic dementias are accompanied by inhibition of pyruvate and ketoglutarate dehydrogenase (PDH, KDH) in affected
brains. Therefore, we investigated how amprolium-evoked thiamine pyrophosphate deficits impair acetyl-CoA metabolism as
well as function and viability of cholinergic neurons. Two day
exposure of differentiated (DC) or nondifferentiated (NC) SN56
cholinergic cells cultured in Minimal Eagles Medium to amprolium caused concentration-dependent inhibition of cell MTT reducing capacity and increased their mortality, that at 5 mM inhibitor concentration reached values 30 and 15% or 43 and 10%,
respectively. However, neither PDH nor KDH activities nor cell
growth were significantly, inhibited by their culture with amprolium. On the other hand, amprolium decreased acetyl-CoA levels
in DC and NC by 39 and 46%, respectively. The inhibitor altered
choline acetyltransferase activity neither in NC nor in DC. Acetylcholine content in DC and was suppressed by amprolium for
over 40%. On the contrary, in NC it had no effect on the transmitter level. These fi ndings indicate that increased ACh synthesis in
DC made them more vulnerable to acetyl-CoA shortages caused
by amprolium-evoked inhibition of thiamine uptake. Low rate of
ACh synthesis in NC, allowed them to save more acetyl-CoA to
support energy metabolism and thereby facilitated their survival
under pathologic conditions. Supported by MNiSW projects PO5A
11030, NN401 2333 33 and AMG fund W-144.
TIII.35
ENERGETIC STATUS OF MITOCHONDRIA IN
PRIMARY CEREBELLAR GRANULE NEURONAL
CULTURES OF RATS PRE- AND POSTNATALLY
EXPOSURED TO LEAD
Baranowska-Bosiacka I.1, Rutkowska M.2, Marchlewicz M.3,
Kolasa A.3, Piotrowska K.3, Gutowska I.1, Slawinski G.4
1
Department of Biochemistry and Medical Chemistry, Pomeranian
Medical University, Szczecin, Poland; 2 Department of Biochemistry,
University of Szczecin, Szczecin, Poland; 3 Department of Histology
and Embryology, 4 Department of Anatomy, Pomeranian Medical
University, Szczecin, Poland
Lead (Pb2+) is a widespread pollutant and potent central neurotoxin. We have studied its effect on energetic metabolism of cerebellar granule cells (CGC) in pre- and postnatally intoxicated
rats. Pregnant Wistar rats received 0.1% lead acetate in water.
CGC were prepared from 8-day-old born rats according to a
standard procedure. Pb2+concentration was measured in blood
and in cerebellum homogenates by AAS. Intracellular Pb2+ concentrations in CGC was studied by fluorescent microscopy, in
neurons loaded with the Ca 2+-sensitive indicator Oregon Green.
Intracellular Pb2+ was evaluated from the fluorescence intensity
and estimate in μM range. Pb-induced decrease in Adenylate
Energy Charge (by 10%) and ATP concentration (by 35%) in
cerebellum homogenates (HPLC method) was observed at Pb
concentrations in whole blood (7.05 ± 2.05 μg/dL).The function
of mitochondria of neurons of Pb-treated and control rats were
evaluated using: Mitotracker Green FM and JC-1. We observed
active mitochondria stained green with Mitotracker in cytoplasm of CGC from control and Pb-treated rats. Incubation of
cultured CGC from rats of control groups with the mitochondrial probe JC-1 produced red-orange fluorescence showing
mitochondria with high membrane potential ΔΨ. The green
fluorescence dependent on mitochondria with low ΔΨ was visible in cytoplasm of cells in Pb-treated rats. Pre- and postnatal
low-level exposure to lead caused disruption of mitochondria
energetic status in neurons.
TIII.36
SELECTIVE METABOTROPIC GLUTAMATE
RECEPTOR 1 ANTAGONIST INHIBITS KAINATEINDUCED EXCITOTOXICITY IN VITRO AND IN VIVO
Domin H.1, Zieba B.1, Lazarewicz J.2, Kajta M.3, Smialowska M.1
1
Department of Neurobiology, Institute of Pharmacology PAS,
Kraków, Poland; 2 Department of Neurochemistry, Mossakowski
Medical Research Centre PAS, Warszawa, Poland; 3 Department of
Experimental Neuroendocrinology, Institute of Pharmacology PAS,
Kraków, Poland
The aim of the study was to investigate a neuroprotective
effect of selective mGluR1 antagonist EMQMCM against
kainate-induced excitotoxicityin vitro in mouse cortical and
hippocampal neurons and in vivo in the rat hippocampus. Excitotoxic damage in the cultures was evoked by incubation with
150 μM kainate (KA) for 24 h (hippocampus) or 48 h (cortex).
In in vivo studies male Wistar rats were unilaterally injected
with KA (2.5 nmol/1 μl) into the CA1 hippocampal region. In
both models EMQMCM was added 30 min, 1, 3 or 6 h after
starting intoxication. In vitro KA neurotoxicity was measured
by lactate dehydrogenase (LDH) efflux from damaged cells
into culture media. The application of EMQMCM (0.1, 1, 10
and 100 μM), 30 min or 1 h after KA, significantly attenuated
the KA-induced LDH release both in cortical and hippocampal
cultures. Protective effects also appeared when the antagonist
was applied 6 h after KA, but only at a concentration 100 μM.
Additionally, EMQMCM prevented KA-induced increase in
caspase-3 activity, which indicated for its antiapoptotic action.
In vivo degeneration was examined by counting of surviving
neurons in CA pyramidal layer in the dorsal hippocampus, using stereological methods. It was found that EMQMCM (5 or
10 nmol/1 μl) given 30 min after KA, significantly attenuated
neuronal damage. The obtained results indicate that EMQMCM can prevent excitotoxic neuronal injury both in vitro and
in vivo studies, soits potential therapeutic role may be suggested.
TIII.37
EFFECT OF LIPOPOLYSACCHARIDE INDUCED
INFLAMATION ON NEUROPEPTIDE Y NEURONS
IN MOUSE HIPPOCAMPUS
Smialowska M.1, Domin H.1, Zieba B.1, Czapski G.2,
Strosznajder J.2
1
Department of Neurobiology, Institute of Pharmacology
PAS, Kraków, Poland; 2 Department of Cellular Signalling,
Neuropeptide Y (NPY), a 36 amino acid neurotransmitter, is
involved in the regulation of emotional behavior and its role in
the central responses to peripheral immune challenge is postulated. Plasma NPY levels rises in human sepsis and NPY improved survival in the experimental endotoxic shock induced
by lipopolysaccharide (LPS) injection. Some of the behavioral
effects of LPS, anorexia, depression and anxiety-like behavior
are also modulated by NPY. Therefore, in the present study tried
to fi nd out if LPS affected NPY neurons in mouse hippocampus, a structure most susceptible to damage and involved in the
regulation of emotion. Male C57BL/6 mice were injected with
LPS (1 m/kg, i.p.) and their brains were taken after 6 or 24 h.
The brains were fi xed with paraformaldehyde, cut into frontal
sections containing the dorsal hippocampus and processed by
immunohistochemistry using an NPY antibody. NPY-immunoreactive neurons were counted stereologically in hippocampal
subregions CA1+2, CA3 and DG+hilus, and results were statistically analysed. It was found that after 24 h LPS decreased by
ca. 38% the number of NPY-positive neurons in the hippocampal CA regions. The effect was particularly significant in the
CA3 area. Moreover, staining intensity was diminished. The
obtained results indicate a decrease in NPY expression in the
hippocampus, which may be due to the peptide release induced
by LPS inflammatory action. Supported by MS&HE.28/E32/
BWSN-0053/2008
318
TIII.38
NEUROPROTECTIVE POTENTIAL OF BIPHALIN,
MULTIRECEPTOR OPIOID PEPTIDE, AGAINST
EXCITOTOXIC INJURY IN VITRO
Adamiak M.1, Kowalczyk J.1, Lipkowski A.2, Zablocka B.1
1
Molecular Biology Unit, 2 Department of Neuropeptide,
Biphalin is a dimeric opioid peptide, that exhibits a high affinity for
all three opioid receptors types (miu, delta and kappa). When administered intracerebroventricularly it has been shown to be more
potent than morphine and ethorphine at eliciting antinociception.
Biphalin is currently undergoing intensive preclinical study. It is
recognized in recent years that activation of delta-opioid receptor
elicits neuroprotection against hypoxia and ischemia, but the underlying mechanisms are not fully understood. In this study we tested
the effect of biphalin and the inhibition of opioid receptors by naltrexon (Ntx) on survival of neurons in rat organotypic hippocampal
culture challenged with NMDA in vitro. Cell survival was estimated with fluorescent cell-death marker propidium iodide. Our
main findings are that (1) biphalin in the concentration of 100 μM
reduces NMDA-induced neuronal damage; (2) biphalin neuroprotective effect is abolished by opioid receptors antagonist naltrexon;
(3) naltrexon by itself does not change NMDA-induced neuronal
damage; (4) reduced number of dead cells was clearly shown even
when biphalin was applied with a delay after NMDA challenge.
These results demonstrate that biphalin, multireceptor opioid peptide, can reduce NMDA-induced neuronal damage. This work was
supported by MSHE Polish Mitochondrial Network grant.
TIII.39
INFLUENCE OF ISOPROPYL
METHYLPHOSPHONOFLUORIDATE INTOXICATION
ON CYTOKINE LEVELS IN CHOSEN BRAIN
STRUCTURES
Kowalczyk M., Zdanowski R., Antkowiak B., Paluch M.
Department of Pharmacology and Toxicology, Military Institute of
Hygiene and Epidemiology, Warszawa, Poland
Poisoning with organophosphate compounds induces pronounced
neurotransmitter disturbances in central nervous system. There
are some data indicating that these poisonings initiate changes in
neuro-immune response. Our study was aimed at the evaluation of
cytokines levels (interleukin 1β – IL-1β, interleukin 2 – IL-2, tumor
necrosis factor α – TNF-α and interferon γ – IFNγ) after isopropyl
methylphosphonofluoridate (IMPF) intoxication in chosen brain
structures of BALB/c mice. Tests were performed in supernatants of
frontal cortex, hippocampus and brain stem homogenates using immunoenzymatic assay after 24 h, 7, 14 and 21 days of IMPF intoxication (1/2 LD50, s.c.). IMPF induced differential changes in cytokines
levels (except IL-2) in the examined brain structures. The significant
increase of TNF-α and IL-1β levels was shown in frontal cortex after
14 days of poisoning. Raised level of TNF-α in this structure was
observed until the end of the experiment (21st day). We also observed
pronounced increase of IFNg level after 7 days in hippocampus and
after 7 and 14 days in brain stem. Concurrently the level of IL-1β in
these structures was significantly decreased, in hippocampus in all
measurements, while in the brain stem only after 24 hours following IMPF application.Our results indicate that IMPF intoxication
without inducing convulsions can produce marked changes in brain
cytokines levels that may lead to immune response disturbances.
TIII.40
CHANGES IN CEREBROSPINAL FLUID
CONCENTRATION OF OXIDATIVE STRESS
MARKERS DURING CHEMOTHERAPY OF ACUTE
LYMPHOBLASTIC LEUKAEMIA IN CHILDREN
Protas P.1, Muszynska-Roslan K.2, Holownia A.1,
Krawczuk-Rybak M.2, Braszko J.1
1
Department of Clinical Pharmacology, 2 Department of Pediatric
Oncology, Medical University of Bialystok, Białystok, Poland
In this study we advised question whether cerebrospinal fluid oxidative stress markers are associated with neurotoxicity of chemotherapy
of acute lymphoblastic leukaemia (ALL). Examination of 38 ALL patients revealed a statistically significant increase in 8-isoprostane and
decrease in total antioxidative capacity during the treatment. Dynamic analysis revealed a statistically significant increase in isoprostane
starting on the 59th day of the treatment when the levels were highest
and remained raised during all the treatment course. The mean 8-isoprostane level at the diagnosis was 9.05 ± 5.12 pg/ml, and no correlation with initial leukocytosis, organomegaly and lactate dehydrogenase level was noted. Dynamic data analysis revealed a statistically
significant increase in 8-isoprostane on the 59th day of the treatment
(24.85 ± 26.28) and at four points during consolidation phase (17.28
± 8.09; 22.72 ± 21.79; 24.92 ± 22.74; 32.32 ± 26.85) as compared to
its level at the diagnosis (P<0.01. The mean total antioxidative capacity level at the diagnosis was (203.98 ± 15.11 μmol/l). Dynamic data
analysis revealed a statistically significant decrease in total antioxidative capacity on the 59th day of the treatment (189.76 ± 4.64) and at one
point during consolidation phase (188.29 ± 8.46) as compared to its
level at the diagnosis (P<0.05). The study suggests that standard ALL
treatment may cause neurotoxicity by oxidative stress.
TIII.41
SYSTEMIC INFLAMMATION LEADS TO
UP-REGULATION OF PROOXIDATIVE GENES AND TO
ACTIVATION OF DEATH SIGNALLING
IN HIPPOCAMPUS
Czapski G.1, Chalimoniuk M.1, Jacewicz M.1, Gajkowska B.2,
Smialowska M.3, Strosznajder J.1
1
Department of Cellular Signalling, 2 Department of Cell
Ultrastructure, Mossakowski Medical Research Centre PAS,
Warszawa, Poland; 3 Department of Neurobiology, Institute of
Our recent data have shown that systemic inflammation evoked
by lipopolysaccharide (LPS) stimulates NO-mediated signalling
pathways in the midbrain. The aim of the present study was to
investigate the dynamics of proinflammatory genes expression in
mouse hippocampus during 4 days after intraperitoneal injection
of LPS (1 mg/kg b.w.). Moreover, the selected biochemical processes involved in cell death pathways were analysed. Real-time
PCR analysis indicated the significant time-dependent alteration
of several prooxidative genes expression. During first 3 h posttreatment the level of mRNA for COX-2, iNOS and TNFα was
increased, then after 6–24 h for cPLA2, TLR4, 5-LOX and 12LOX, respectively. Immunohistochemical analysis showed an appearance of many activated microglia cells, confirming activation
of local inflammatory reaction in hippocampus. The biochemical
and molecular studies demonstrated the significant activation of
apoptosis inducing factor (AIF)-mediated, caspase-3 -independent
death signalling and cathepsin B-related autophagy. These data
were confirmed by electron-microscopic examination of cell ultrastructure. The presented genetic, biochemical and morphologic alterations induced by systemic inflammation could be responsible
for impairment of learning and memory. This study was supported
by Scientific Network 28/E-32/BWSN-0053/2008.
TIII.42
MANIPULATION OF Ttyh1 GENE EXPRESSION
INFLUENCES CELL MORPHOLOGY IN
NEUROBLASTOMA AND HIPPOCAMPAL
NEURONS IN VITRO
Stefaniuk M., Lukasiuk K.
Ttyh1 gene is a member of tweety family of putative large conductance maxi-Cl- channels. According to our previous data, expression
of Ttyh1 mRNA in the brain is localized in neurons and is increased
following status epilepticus in the animal model of epileptogenesis.
The function of Ttyh1 has not been elucidated. Here we aimed at
characterization of Ttyh1 by overexpressing or silencing it in neuroblastoma cell line or in hippocampal neurons in vitro. In hippocampal neurons transfected with plasmid coding Ttyh1-EGFP
fused protein, Ttyh1-EGFP was present in dots along the neurites
and at the ends of new formed projections. Similar Ttyh1-EGFP localization was observed in neuroblastoma cells. Overexpression of
Ttyh1 in hippocampal neurons in vitro induced formation of new,
often branched projections as soon as after 24 h. Even more intense
branching was observed when cells were transfected with Ttyh1 7 or
14DIV. Ttyh1 silencing in hippocampal neurons in vitro using siRNA increased the number and length of primary dendrites. In addition, it affected cell morphology causing abnormal pattern of MAP2
distribution. Since manipulation of Ttyh1 expression influences cell
morphology and distribution of cytoskeleton elements, we propose
that Ttyh1 is involved in cytoskeleton functions. It is tempting to
suggest, that by influencing the neurite growth and ramification,
Ttyh1 participates in aberrant network formation and by this – the
development of epilepsy. Supported by MNiSW N N301 162135
TIII.43
c-Fos MAPPING OF CHANGES IN NEURONAL
ACTIVITY DURING THE COURSE OF PTZ KINDLING
OF SEIZURES
Szyndler J.1, Maciejak P.2, Turzynska D.2, Sobolewska A.2,
Taracha E.2, Skorzewska A.2, Lehner M.2, Bidzinski A.2,
Plaznik A.2
1
Department of Experimental and Clinical Pharmacology,
Medical University of Warsaw, Warszawa, Poland; 2 Department of
Neurochemistry, Institute of Psychiatry and Neurology, Warszawa,
Poland
In the present study, the c-Fos expression was used to map brain
structures recruited during the evolution of seizures after repeated,
administration of pentylenetetrazol at the subconvlusive dose (35
mg/kg, i.p.), in rats. It has been found that the earliest expression of cFos, at the stage 1,2 of kindling, appeared in the nucleus accumbensshell, the piriform cortex, the prefrontal cortex and striatum. On the
3rd stage of kindling, the central amygdala nucleus, the entorhinal
cortex, and the lateral septal nucleus (LSV), showed an enhanced expression of c-Fos. On the 4th stage of kindling, c-Fos was increased in
the basolateral amygdala, and CA1 area of the hippocampus. Finally,
c-Fos labelling was enhanced in the dentate gyrus of the hippocampus, only when the stage 5 of kindling, i.e. the clonic-tonic convulsions, appeared. The most potent changes in c-Fos (in a descending
order) were shown in the dentate gyrus, piriform cortex, CA1 area,
the LSV, basolateral amygdala, central amygdala nuclei, and prefrontal cortex. It appeared, that there are important similarities in the
structures recruited at the beginning and at the end of electrically
and chemically-induced kindling, i.e. the piriform cortex and the hippocampal dentate gyrus, respectively. On the other hand, the differences gradually disappear at the later stages of kindling, followed
by the symmetrical propagation of epileptic activity from the limbic
system to the neocortex, during the generalized seizures.
TIII.44
A NOVEL METHOD TO VISUALIZE TISSUE
MOLECULAR ARCHITECTURE AT THE LEVEL
OF SYNAPSE
Szepesi Z.1, Szczepankiewicz, A.2, Walczak A.2, Wilczynski G.2,
Wlodarczyk J.1, Kaczmarek L.1
1
of Neurophysiology, Nencki Institute of Experimental Biology PAS,
Warszawa, Poland
To understand various biological functions of CNS, visualization
of the fine details of tissue molecular architectures and synaptic
structures is of the major importance. Herein, we use a method for
immunfluorescence microscopy called Array Tomography which
allows for imaging details of tissue structure. To this end, we cut
resin-embedded specimen into serial sections (50–250 nm) by ultramicrotome. Hydrophilic resin permits the antibody penetration into
the tissue, thus providing an opportunity for post-embedding immunostaining. The ribbon of serial sections represents tissue volume. Reconstruction of three dimensional distributions of antigens
is obtained by imaging and post-processing of consecutive images.
The array can be eluted and re-stained, thus allowing for detection
of several antigens in the same sample. The above methodology provides visualization at the spatial resolution that is higher than the
one obtained using conventional confocal microscopy. Furthermore,
we use this method together with the super-resolution (70 nm) stimulated emission depletion (STED) microscopy. Thus, combining of
these two methods provides very detailed image of tissue architecture and offers a novel opportunity to reconstruct the molecular architecture with sub-micrometer resolution in three dimensions.
TIII.45
NITRIC OXIDE SYNTHESIS INHIBITOR INCREASES
SEVERITY OF HOMOCYSTEINE- INDUCED SEIZURES
IN ADULT RATS
Hrncic D.1, Rasic-Markovic A.1, Susic V.2, Djuric D.1,
Stanojlovic O.1
1
Laboratory of Neurophysiology, Institute of Medical Physiology
Richard Burian, Belgrade University School of Medicine, Belgrade,
Serbia; 2 Department of Medical Sciences SASA, Belgrade, Serbia
Homocysteine, endogenous sulphur – containing amino acid, displays neuroexcitatory effects and acts as a convulsant agent by still
320
unclear mechanisms. Reported results on the role of nitric oxide
(NO) in epileptogenesis are highly contradictory. NO levels could be
decreased by N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of enzyme NO synthase (NOS). The aim of the current study was
to examine the effects of L-NAME on seizure susceptibility induced
by subconvulsive dose of D,L homocysteine – thiolactone (HCT) in
rats. Adult male Wistar albino rats were intraperitoneally (i.p.) treated with HCT 5.5 mmol/kg and observed for seizure behavioral manifestations during next 90 min. Increasing doses of L-NAME (200,
500 and 700 mg/kg, i.p., n=6, 7 and 8, respectively) or saline (n=9)
were injected 30 min prior to HCT administration. Seizure episode
severity was assessed using descriptive rating scale with four grades.
L-NAME in doses of 500 and 700 mg/kg significantly increased severity of homocysteine seizures (P<0.05) when compared with saline –injected rats. The lowest applied dose of L-NAME (200 mg/kg)
had no statistically significant effect on severity of seizures induced
by HCT. Based on these results it cold be concluded that L-NAME,
NOS inhibitor, potentiates convulsive properties of HCT.
TIII.46
PERFUSION SENSITIVE CONTRAST ENHANCED
MR IMAGING OF DYSEMBRYOPLASTIC
NEUROEPITHELIAL TUMOR (DNT):
A NEW NEUROIMAGING FINDING
Mazioti A.1, Markoni A.2, Wozniak G.1, Lavdas E.1, Vassiou K.1,
Fezoulidis I.1
1
University Hospital of Larissa, Larissa, Greece; 2 EuroMedica
Diagnostic Center Radiology Department, Larissa, Greece
Dysembryoplastic neuroepithelial tumors (DNTs) are benign lesions affecting young people and are associated with epilepsy.
There have been described more than 300 cases in the literature
and the clinical, pathologic and radiological findings are well
known. Recent advances in neuroimaging allow the acquisition
of cerebral microcirculation parameters by perfusion weighted
imaging giving additional diagnostic information improving the
diagnostic accuracy. The aim of this study is to show the Perfusion sensitive contrast enhanced MR imaging findings of a case of
DNT as an additional neuroradiological finding. Further investigation of microcirculation parameters may be helpful to perform the
correct diagnosis of such tumors.
TIII.47
SEIZURES CONTROL IN FREE-RUNNING RATS
Smyk M.1, van Luijtelaar G.2, Coenen A.2, Lewandowski M.1
1
Department of Neurophysiology and Chronobiology, Jagiellonian
University, Kraków, Poland; 2 Donders Centre for Cognition, Donders
Institute for Brain, Cognition and Behaviour, Radboud University,
Nijmegan, The Nederlands
Absence epilepsy is characterized by disturbed consciousness and
generalized, synchronous, bilateral, 3–4 Hz (in humans) and 7–11
(in rats) spike-wave discharges (SWD) in EEG. It has been documented that the occurrence of absence seizures shows circadian
pattern both in humans and animals. WAG/Rij rats, a well known,
validated animal model of human absence epilepsy, show a clear
circadian distribution of SWD (maximum in early hours of dark
period, minimum after the onset of light). Moreover, a strong correlation between the occurrence of SWD and the level of vigilance exists: SWD are less prone to occur during active wakefulness. SWD
rhythm and its relationship with activity in entrained and constant
condition were investigated. Chronic EEG and general activity recordings were made in six adult WAG/Rij rats. Animals were kept
in 12:12 light–dark cycle. The light regime was changed after 10
days into constant dim light (<6 lux) in which rats were maintained
for the following 20 days. The period lengths of both rhythms were
estimated by the Cosinor method. Clear 24 h rhythms of activity
and SWD were found under entrained conditions, in constant condition both rhythms were free-running. Periods’ length of activity
was increased while period of SWD rhythm was changed differently for different animals. Observed rhythms’ splitting suggests
decoupling of these two rhythms in constant condition and that the
SWD rhythm is no longer controlled by a master clock.
TIII.48
EEG CLUSTERIZATION PATTERNS IN DIFFERENT
EXPERIMENTAL CONDITIONS
Levin E.1, Savostyanov A.1, Tsai A.2, Lee J.2, Knyazev G.1
1
Laboratory of memory regulation mechanisms, State Research
Institute of Physiology SB RAMS, Novosibirsk, Russia; 2 Institute
of Statistical Science, Academia Sinica, Taipei, Taiwan
EEG data, obtained in 3 groups of young (18–30 years) subjects
during different experimental conditions (background brain activity with open/closed eyes, emotional face recognition and
stop-signal task) were analyzed. Event-related desynchronization
(ERD) and event-related spectral perturbations (ERSP) were used
as functional response measures. Individual cortical distributions
of these responses were compared for different modalities. Group
1 (32 women, 16 men) performed stop-signal task, group 2 (21
women, 19 men) – facial expression recognition task, and group
3 (2 women, 13 men) both of these tasks. Background EEG with
open and closed eyes was recorded in all three groups. EEGs for
groups 1 and 2 were recorded using 32-channel “Neurovisor-24”
amplifier, and for group 3- by 132-channel “Neuroscan” amplifier.
Event-related reactions were obtained using ERD for group 1 and
ERSP for groups 2 and 3. Percentage change was used as measure
of reaction on opening eyes. Electrodes were grouped into clusters
with similar “behavior” separately for each condition using factor
analysis with principal components extraction and varimax rotation. Number of Identified clusters increased and size of clusters
decreased with increase of analyzed frequency. Importantly, clusters for each frequency (especially for lower ones) were similar
between different conditions, but differed between subjects, supporting hypothesis of stability of brain oscillatory systems’ spatial
characteristics.
TIII.49
PREDICTION OF PROTEIN SECONDARY STRUCTURE
IN BRAIN TISSUES AND MEMBRANES USING NEURAL
NETWORKS BASED ON FTIR SPECTROSCOPY
Severcan M.1, Akkas S.2, Cakmak G.2, Turker Gorgulu S.2,
Severcan F.2
1
Department of Electrical and Electronic Engineering, 2 Department
of Biological Sciences, Middle East Technical University, Ankara,
Turkey
Membrane and tissue proteins in biological systems are difficult
to be isolated and crystallized. In recent years, a low resolution
technique, Fourier Transform Infrared (FTIR) spectroscopy, has
been improved and proven to be a reliable tool to determine the
secondary structure of proteins. Using this technique, it is possible
to see protein bands directly from the spectra of tissues and membranes which allow us to obtain information about protein secondary structure. Generally the information is deduced by monitoring
the amide I band, which is located between 1700–1600 cm-1. This
band is the result of many overlapping individual peaks arising
from different protein conformations, such as α-helix, β-sheet or
turns. In the current study, using Neural Network (NN) method
based on FTIR spectra [Severcan et al. (2004) Anal Biochem], the
radiation, epilepsy and antioxidant-induced variations in the secondary structure of proteins in brain tissues and membranes were
determined. The results showed that lipoic acid treatment revealed
an unaltered protein secondary structure, while radiation caused
a significant increase in random coil structure and epilepsy led to
a significant decrease in β-sheet structure in rat brain tissue. This
study confirms that NN approach based on FTIR data is a fast and
effective method to predict the secondary structure of proteins in
different biological systems.
TIII.50
A TEST OF WHETHER N-ACETYL-ASPARTYLGLUTAMATE AND N-ACETYL-ASPARTATE CAUSE
DAMAGE IN LEUKODYSTROPHIES BY ACTIVATING
OLIGODENDROCYTE NMDA RECEPTORS
Kolodziejczyk K., Hamilton N., Wade A., Karadottir R.,
Attwell D.
Department of Neuroscience, Physiology and Pharmacology,
University College London, London, UK
Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG)
and N-acetyl-aspartate (NAA) are associated with myelin loss in
the leukodystophies Canavan’s disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate neuronal NMDA receptors, and also act on group II mGluRs. Since NMDA receptors
are present on oligodendrocytes [Káradóttir et al. (2005) Nature
438: 1162], we hypothesised that NAA and NAAG may damage
oligodendrocytes by activating their NMDA receptors, causing
a deleterious Ca2+ influx. We show that NAAG, but not NAA,
evoked an inward membrane current in cerebellar white matter
oligodendrocytes, which was reduced by NMDA receptor block
(but not by block of mGluRs). The size of the current evoked by
NAAG, relative to that evoked by NMDA, was much smaller in
oligodendrocytes than in neurons, and NAAG induced a rise in
[Ca2+]i in neurons but not in oligodendrocytes. In addition, a major
part of the response in oligodendrocytes, but not in neurons, was
blocked by TTX. We conclude that the NAAG-evoked current in
oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors and is unlikely to be a major contributor
to white matter damage in the leukodystrophies. Supported by the
Wellcome Trust and Royal Society.
TIII.51
STREPTOZOTOCIN INDUCED DIABETES CHANGES
CONDUCTION VELOCITY DISTRIBUTION OF RAT
SCIATIC NERVE IN A TIME DEPENDENT MANNER
Tuncer S., Dalkilic N.
Department of Biophysics, Selcuk University Meram Medical
Faculty, Konya, Turkey
Diabetes is a metabolic disorder based on uncontrolled blood
glucose concentration that affects majority of human population. This disorder causes some pathologies in nervous system
that called diabetic neuropathies. As a secondary complication
of diabetes, diabetic neuropathies causes damage on peripheral
nerves depending on time of diabetes. In this study, experimental type 1 diabetes was induced by injection of streptozotocin (50 mg/kg, i.p.) in rats. Diabetic animals were grouped as
2 and 4 weeks diabetic. Control group animals were received
only vehicle of STZ (0.1 M Citrate, pH 4.5). Sciatic nerves of
experimental animals were dissected and electrophysiological
recording experiments were achieved. Recorded compound action potentials were then analyzed to understand the time dependent effects of diabetes on peripheral nerves. Analysis have
shown that, current recording techniques that is used in clinics for diagnoses of neuropathologies, carries less information
compared to conduction velocity distribution (CVD) histograms
obtained by using single fiber action potential (SFAP) models.
In parameters obtained by using conventional methods, statistically significant changes were fi rst seen in 4th week of diabetes.
But, by means of CVD histograms, diabetes induced significant
changes could be seen 2nd week of diabetes in slowly conducting
fiber group. So, CVD methods give more information in early
diagnosis of neuropathies compared to classical conduction velocity measurements.
TIII.52
AUTOIMMUNE ENCEPHALOMYELITIS IS
ACCOMPANIED BY THE INJURY OF HIPPOCAMPAL
NEURONS
Kurkowska-Jastrzebska I.1, Zaremba M.2, Cudna A.1, Piechal A.2,
Swiatkiewicz M.2, Zaremba M.3, Czlonkowska A.1,2,
Oderfeld-Nowak B.3
1
2nd Department of Neurology, Institute of Psychiatry and
Neurology, Warszawa, Poland; 2 Department of Experimental and
Clinical Pharmacology, Medical University of Warsaw, Warszawa,
Poland; 3 Department of Molecular and Cellular Neurobiology,
Multiple sclerosis (MS) is associated with cognitive deficits,
developing independently from motor disorders. These deficits
may be associated with brain neuronal damage. In this study,
using the experimental model for MS – autoimmune encephalomyelitis (EAE), we investigated whether EAE will result in the
damage of hippocampal neurons and selective deficits in learning and memory, and whether there may be a correlation between
the two phenomena. Lewis rats 3 months old were injected with
4 millions of anti-MBP CD4+ T cells to evoke EAE. Animals
suffered from tail and hind limb paresis and recovered after 10
dpi. T cells infiltrated spinal cord and many brain regions including hippocampus. We demonstrated the decrease of pyramidal
neurons in CA4 region by about 20%, as evaluated by stereological measurements, at 21 dpi. This was preceded by prolonged
glial activation as well as by a rise of the pro-inflammatory cytokine mRNA expression (IL-1β, IL-6 and TNF α). However,
no differences in the water maze test were detected between the
EAE and control groups, on 21 dpi and on 90 dpi. In conclusion,
anti-MBP CD4+ T cells are capable of injuring hippocampal pyramidal neurons during EAE, probably, through the secretion
of pro-inflammatory cytokines. However, in the studied conditions, hippocampal neurodegeneration caused by T cells did not
result in memory disturbances. Supported by the grant no N4011293-33 of the Ministry of Scientific Research and Information
Technology in Poland.
322
TIII.53
ASTROGLIAL REACTION IN THE EARLY
ASYMPTOMATIC PHASE OF EXPERIMENTAL
AUTOIMMUNE ENCEPHALOMYELITIS
Grygorowicz T.1, Sulkowski G.1, Sulejczak D.2, Lenkiewicz A.1,
Struzynska L.1
1
Department of Neurochemistry, 2 Department of Experimental
Pharmacology, Mossakowski Medical Research Centre PAS,
Warszawa, Poland
Under different pathological conditions activation of astrocytes of
neuroprotective or neurotoxic nature is observed. There is a growing number of evidence that many pathological states of brain
are characterized by very early active contribution of astrocytes
to neurodegenerative axonal damage. Astroglia posses defense
mechanisms against glutamate excitotoxicity (transporter systems)
but may also contribute to the enhanced release of this potentially
toxic amino acid trough exocytosis, P2X7 purinergic receptors,
hemichannels or reversing of glutamate transporters. These cells
are also a main source of ATP, active signaling molecule, which
activates many purinergic receptors in brain, including P2X7R,
which participates in development of inflammation and neurodegeneration phenomena. The aim of this study was to investigate
the expression of astroglia-specific proteins during the course of
EAE using immunochemical and immunohistochemical analysis.
We observed early activation of astroglia in the inductive phase
of EAE (4 day p.i.) which was connected with overexpression of
GFAP and S-100β. Expression of Cx43, protein that forms hemichannels was also enhanced so as the expression of P2X7R. Additionally, the level of GLT-1 glutamate transporter’s protein increase
significantly. The results suggest that in EAE pathology very early
activation of astroglia takes place in the preclinical stage of the
disease. The exact nature of this activation will be investigated.
TIII.54
THE 8 kD FORM ENCODED BY THE HTLV-1 ORF-I
IS ASSOCIATED WITH LOW PROVIRUS LEVELS
AND HEALTHY CARRIER STATE IN A SUBSET
OF HTLV-1 INFECTED INDIVIDUALS
Bialuk I.1,2, Walser J. 3, Fukumoto R.1, Andresen V.1,
Graham J.4, Jacobson S.4, Gessain A.5, Alcantara L.6,
Galvao-Castro B.6, Franchini G.1
1
Animal Models and Retroviral Vaccines Section, National
Cancer Institute, Bethesda, USA; 2 Department of General
and Experimental Pathology, Medical University of Bialystok,
Białystok, Poland; 3 Section on Genomic Structure and Function,
Laboratory of Molecular and Cellular Biology, National Institute
of Diabetes and Digestive and Kidney Diseases, Bethesda, USA;
4
Viral Immunology Section, National Institute of Neurological
Disorders and Stroke, Bethesda, USA; 5 Oncogenic Virus
Epidemiology and Physiopathology Unit, Department of Virology,
Pasteur Institute, Paris, France; 6 Oswaldo Cruz Foundation
Salvador, Bahia, Brazil
HTLV-1 infection is linked with a neurodegenerative disorder HTLV1 associated myelopathy/tropical spastic paraparesis (HAM/TSP).
Expression of the singly or doubly spliced ORF-I cDNA of HTLV-1
results in the production of the 12 kD and 8 kD protein isoforms.
The uncleaved 12 kD form resides in the ER and affects MHC-I
and IL-2R. The cleaved 8 kD form traffics to the cell surface, is recruited to the immunological synapse upon T-Cell Receptor (TCR)
stimulation decreasing TCR signaling and viral replication. Genetic
analysis of ORF-I from ex vivo samples of HTLV-1 infected individuals reveals amino acid substitutions that affect its proteolytic
cleavage, suggesting that ER or membrane associated functions of
ORF-I may contribute to the persistence of HTLV-1 infected T-cells
in the host. To investigate a putative relationship between ORF-I
forms and provirus level, the best predictor of disease development,
we measured the provirus level in the blood and linked it with the
presence of ORF-I isoforms. The provirus levels ranged from 0.2
to 165 copies of proviral DNA per 106 PBMCs. DNA sequencing
and reverse genetics revealed mutations at/in the vicinity of both
cleavage sites within ORF-I. A rare mutation found at position 26 in
ORF-I resulted mainly in the presence of the 8 kD isoform. Importantly, patients with this mutation had significantly lower virus that
those that carried either 12 kD isoform or both forms, and with one
exception they belonged to a healthy carrier group.
TIII.55
METABOLOMIC ANALYSIS AND SIGNATURES IN
AMYOTROPHIC LATERAL SCLEROSIS: 1H NMR
STUDIES OF SERUM
Kumar A., Kalita J., Misra U., Babu G.
Department of Neurology, Sanjay Gandhi Postgradute Institute
of Medical Sciences, Lucknow, India
Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disease characterized by progressive dropout of motor neurons
in the brain, brain stem, and spinal cord. This disease may produce characteristic perturbations of the metabolome, the collection
of small-molecules (metabolites). To test this hypothesis, we have
investigated metabolite profile in the blood serum of 30 patients
with ALS and 25 healthy controls by using 1H NMR. Patients with
ALS had significantly higher median concentrations (microM)
of creatine/creatinine (43 vs. 30, P<0.02), glutamate (92 vs. ND,
P<0.001), β-hydroxybutyrate (2 vs. ND, P<0.001), acetate (15 vs.
8, P<0.01), acetone (15 vs. 9, P<0.05), and formate (16 vs. ND,
P<0.001) than healthy controls, and significantly lower concentrations of N-acetyl derivatives (P<0.001), glutamine (406 vs. 448,
P<0.02), histidine (55 vs. 67, P<0.001), however, the concentration of alanine, lysine, pyruvate, citrate, glucose, and tyrosine were
comparable. Concentration of glutamate, N-acetyl derivatives and
histidine correlated with the duration of the disease. These results
suggest that metabolomic studies can be used to ascertain metabolic
signatures of disease in a single step. Such 1H NMR study of serum
provides an insight into aberrant biochemical pathways which may
have the potential to serve as a surrogate marker for monitoring
ALS disease progression and also could be targets for drug design.
TIV: Sensory and Motor Systems
TIV.01
M100-PEAK AMPLITUDES OF DIFFERENT SUBJECTS,
HEMISPHERES, AND STIMULUS CONDITIONS DIFFER
BY FACTORS RATHER THAN BY AMOUNTS
Zacharias N.1, König R.1, Sieluzycki C.1, Heil P.2
1
Special Lab Non-Invasive Brain Imaging, 2 Department of Auditory
Learning and Speech, Leibniz Institute for Neurobiology, Magdeburg,
Germany
The analysis of MEG data commonly involves arithmetic averaging of the evoked magnetic fields (AEFs) across subjects. A tacit
assumption of this practise is that signals from different subjects
simply differ by some amount. If true, the standard deviation (SD)
of signal strengths across subjects should be independent of the
mean signal amplitude. We exploit the dependence of the M100peak amplitude on the stimulus onset interval (SOI) to scrutinize
the variation of SD with mean amplitude. Fifteen subjects were
stimulated monaurally with tones of SOIs varying between 250 ms
and 12.5 s. For each subject and hemisphere, the M100-peak amplitude was determined, either from the channel with the largest
signal or from a cluster of five channels whose signals were averaged arithmetically or geometrically. Irrespective of the measure,
we found that the SD of the M100-peak amplitude across subjects
scales linearly with the amplitude of the corresponding arithmetic
mean, but remains practically constant for the amplitude of the
geometric mean. This shows that the M100 amplitudes of different
subjects, hemispheres, and stimulus conditions differ by factors
rather than by amounts. We also show that the geometric mean
of the M100 amplitudes across different conditions provides an
excellent measure for normalizing such data. Our results question
the common practise of arithmetic averaging across subjects, or
subtraction procedures between conditions, and thus are likely to
have widespread implications.
TIV.02
THE EFFECT OF POSTNATAL BRIEF NOISE
EXPOSURE ON SOUND LEVEL PROCESSING IN
INFERIOR COLLICULUS NEURONS OF ADULT RATS
Grecova J., Bures Z., Popelar J., Suta D., Syka J.
Department of Auditory Neuroscience, Institute of Experimental
Medicine, AV CR, Prague, Czech Republic
The development of the central auditory system is an activitydependent process. Hence, any manipulation of the hearing organ
that temporarily alters the input to the central auditory nuclei can
have a serious and mostly permanent impact on their structure and
function. Our previous study showed that adult rats exposed to
intense brief noise as juvenile had significantly deteriorated frequency selectivity of the inferior colliculus neurons. In this study,
we focused on sound level processing in inferior colliculus neurons in rats exposed for 8 min to intense broad-band noise on
postnatal day 14. At the age of 3–6 months, the neuronal activity in the central nucleus of the inferior colliculus was recorded
under ketamine-xylazine anaesthesia, and the parameters of the
rate-level functions were compared between the neurons of noisetreated rats (n=116) and age-matched controls (n=94). While the
neuronal thresholds in the two groups did not differ significantly,
the neurons of the exposed animals exhibited a significantly short-
er dynamic range and a steeper slope of the rate-level function
compared with controls. The results indicate that a short-lasting
exposure to intense sound during the sensitive period of postnatal
development disrupts the normal coding of sound intensity in the
inferior colliculus neurons. Supported by grants AV0Z50390512,
GACR 309/07/1336, GACR 309/08/H079, IGA NR 8113-4 and LC
554.
TIV.03
ALTERNATIVE SPLICING OF L7/pcp2 GENE RESULTS
IN TRUNCATED PROTEIN
Barski J.1, Lauth M.2, Fertala A.3
1
Department of Physiology, Medical University of Silesia, Katowice,
Poland; 2 Department of Neuroscience and Nutrition, Karolinska
Intitutet, Stockholm, Sweden; 3 Department of Dermatology
& Cutaneous Biology, Thomas Jefferson University,
Philadelphia, USA
The L7/pcp2 have been described for the first time in 1988 by two
independent groups. Transcripted RNA is source of 99-aminoacid peptide of 16kD with some homology to NH2-terminal part
of PDGF. pcp2 protein is known for its very specific expression
in cerebellar Purkinje cells and bipolar retinal neurons. Initial experiments showed, that isolated Purkinje cell specific mRNA did
not hybridize to cerebellar mRNA from mice with Purkinje cell
degeneration mutation. It was a strong indication, that pcp2 protein could play an important role in Purkinje cell physiology and
development. Mice null mutant for that gene however, do not display any morphological or physiological abnormalities. Up to now
there are only few experimental data suggesting the function of
pcp2 protein. Experiments on G protein interactions revealed pcp2
as possible GDP exchange factor for Go subunit. This function
of pcp2 depends on the presence of G-protein regulatory motifs
(GPR) known also as GoLoco motifs. Our experiments regarding
expression pattern of pcp2, revealed a new splice variant of its
mRNA – pcp2B. Additional ~350bp long band was always prominent in PCR products amplified on cDNA from mouse eye preparations. Incorporation of the new exon 3B results in truncated
protein, because of STOP codon present in its structure. Missing
region consists of some putative phosphorylation sites with possible biological function important the L7/pcp2 protein.
TIV.04
SIGNAL FREQUENCY TRANSFORMATION
BY HODGKIN-HUXLEY NEURONS
Borkowski L.
Quantum Physics Division, Faculty of Physics, Adam Mickiewicz
University, Poznań, Poland
The response of a Hodgkin-Huxley neuron strongly depends on
the form of input current. We study the model with a periodic postsynaptic current, where each of the current pulses has the form
I(t) ~ gsyn ∑ (t/τ) exp(-t/τ) Θ(t) (Va-Vsyn), where gsyn is the synapse
conductivity, τ is the time constant associated with the synapse
conduction, Va is the maximum membrane potential and Vsyn is the
reversal potential of the synapse. The other parameters are typical for the Hodgkin-Huxley model. There are three resonant frequencies at 57 Hz, 28.5 Hz, and 19 Hz, where the 57 Hz feature
is the main resonance of the neuron. In the resonant regime the
system has the tendency to mode locking with high values of k,
where k=To/Ti is the ratio of the output ISI to the input ISI. Cha-
324
otic states are present in many areas of the resonant regime. The
mode-locked states within the resonance may have large values of
k. The incoming signal frequency may be substantially reduced
when passing through such neuron. A chain of two or more neurons may decrease the signal frequency by more than an order of
magnitude. Noise in the input signal lowers the response threshold
and improves the signal-to-noise ratio. Network effects are also
discussed. Acknowledgements: Part of the numerical computation
was performed in the Computer Center of the Tri-city Academic
Computer Network in Gdansk, Poland.
TIV.05
MELANOPSIN-BASED PHOTORECEPTION:
CONTRIBUTION TO THE MECHANISM OF
GENERATION OF INFRASLOW RHYTHM
IN THE RAT OLIVARY PRETECTAL NUCLEUS
Orlowska P., Tokarczyk E., Szkudlarek H., Lewandowski M.
University, Kraków, Poland
In mammals, environmental light signals are captured by the
eye’s photoreceptors: rods, cones and intrinsically photosensitive melanopsin retinal ganglion cells (ipRGCs). During the last
few years, ipRGCs were extensively studied and their membrane
properties, projections and physiological role in regulation of circadian rhythms and pupillary light reflex were documented. However, these studies do not explain how activity of ipRGCs affects
physiology of target cells in suprachiasmatic nuclei (SCN), intergeniculate leaflet (IGL) or olivary pretectal nucleus (OPN). Some
of the neurons constituting above mentioned structures, express
slow oscillatory activities that are modulated by light and depend
on functional input from the retina. This implicates, that mechanism of expression of slow oscillation may include synaptic drive
from ipRGCs. Recently, 2-aminoethoxydiphenylborane (2-APB)
was described as an acute inhibitor of ipRGCs activity. This study
combines intravitreous injections of 2-APB with extracellular recordings from oscillatory OPN neurons in urethane anesthetized
Wistar rat. The experiments showed that inactivation of ipRGCs
activity abolish oscillatory pattern and reduce firing rate of OPN
neurons. Injection of comparable volume of physiological saline
in control experiments had no effect on oscillatory activity. To our
knowledge, this is the first study that directly links extraretinal
neuronal firing with activity of ipRGCs.
TIV.06
CORTICAL FEEDBACK MODULATES TEMPORAL
PATTERN OF SPONTANEOUS NEURONAL ACTIVITY
IN THE CAT’S DORSAL LATERAL GENICULATE AND
PERIGENICULATE NUCLEI
Radzikowska Z., Waleszczyk W., Bekisz M., Wrobel A.
In cats with pretrigeminal brainstem transections, reversible inactivation of the primary visual cortex by cooling results in changes
of the magnitude of response and spatio-temporal structure of receptive fields of neurons from lateral geniculate and perigeniculate
nuclei (LGNd and PGN; Waleszczyk et al. 2005). Since cortical
inactivation changes also the spontaneous activity of these thalamic neurons, we hypothesized that cortical feedback affects their
membrane potential. Both types of investigated cells displayed two
modes of activity: a tonic mode, during depolarization; and a burst
mode, when the cell is hyperpolarized. In this report we investigated
the effect of elimination of cortical feedback by cooling on the temporal pattern of the spontaneous "bursty" activity of single neurons
in LGNd and PGN. During such reversible inactivation of areas
17 and 18, in both LGNd and PGN cells, spontaneously occurring
bursts exhibited longer interspike intervals (ISIs) and lower number
of spikes, while the average burst duration remained unchanged.
Longer ISIs suggest that cortical feedback influences not only visual responsiveness of thalamic neurons, but also temporal pattern
of their spontaneous firing, in line with the hypothesis of a tonic
cortical modulation of their membrane potential. Supported by the
Ministry of Science and Higher Education grant COST/127/2007.
TIV.07
A NEW METHOD FOR IDENTIFICATION OF
MODULATION IN NEURAL RESPONSES TO DRIFTING
GRATING STIMULATION
Wypych M.1, Nagy A.2, Paroczy Z.2, Márkus Z.2, Berényi A.2,
Benedek G.2, Waleszczyk W.1
1
Department of Neurophysiology , Nencki Institute of Experimental
Biology PAS, Warszawa, Poland; 2 Department of Physiology, Faculty
of Medicine, Albert Szent-Györgyi Medical and Pharmaceutical
Center, University of Szeged, Hungary
Temporal modulation of responses to drifting grating stimulation is
observed in visual neurons in different brain structures. The common measure of intensity of such modulation is the modulation index
(MI; Movshon et al. 1978), defined as the ratio of the amplitude of
the response component at the stimulus temporal frequency ( f1 ) and
the net response of the cell. However MI works correctly in a limited
range of net responses. If stimulation causes only a weak change
in mean firing rate, MI can take any value independently of actual
modulation in the response. Here we present a new, simple method
of determining the strength of modulation, based on the detection of
a peak in an amplitude spectrum at the frequency of stimulation. We
define modulation strength (MS) as the ratio of f1 value above the
mean value of amplitude spectrum and standard deviation (SD) of
amplitude values along all frequencies in the spectrum. We assume
the response to be modulated if f1 value exceeds the mean amplitude
by at least one SD (MS>1). We test the method on data recorded
from a number of structures of the extrageniculate visual pathway
and compare it to the classical MI and the modulation depth (measure used in the amplitude modulation radio transmission – here
the change of the spectrum component at the stimulation frequency
between the spontaneous activity and the response). Supported by
MNiSW grants: N303 070234, COST/127/2007, N303 046 31/1483,
OTKA/NKTH Hungary grant 68594, OTKA grant PD75156.
TIV.08
MOTOR IMAGERY MEASURED BY FUNCTIONAL
NEAR-INFRARED SPECTROSCOPY (fNIRS)
Holper L.1, Biallas M.1, Eng K.2, Wolf M.1
1
Biomedical Optics Research Laboratory BORL, University
of Zurich, Zurich, Germany; 2 Institute of Neuroinformatics,
University of Zurich and ETH Zurich, Zurich, Germany
Functional near-infrared spectroscopy (fNIRS) is a non-invasive
technique that utilizes light in the near-infrared (NIR) range to determine the oxygenation of localized regions of the brain. The proposed
study aims to establish fNIRS as a valid tool for use in brain-comput-
er interfaces (BCI). Specifically we will use multi-channel fNIRS to
provide feedback of neural activity in a virtual reality rehabilitation
system. BCIs are feedback systems that allow human brain signals to
control devices in real time. fNIRS instruments are non-invasive and
easily adapted to wireless or portable applications, offering decisive
advantages for the development of BCIs compared to fMRI or EEG.
Our project focuses on the development of novel BCI feedback systems based on NIR technology that, in the longer term, will be used
in conjunction with a training systems for the rehabilitation of motor
function. We present results of mental performance of motor imagery (MI) in response to no, positive and negative feedback conditions
indicating that there exist distinct activation pattern concerning occurrence, amplitude and timing. Although, limitations of the current
study require further evaluation, these parameters could be utilized
as a basis to develop pattern classifiers for fNIRS controlled BCIs.
TIV.09
EFFECTS OF PRISM ADAPTION ON VOLUNTARY
AND REFLEXIVE ORIENTING OF ATTENTION
Asanowicz D., Michalczyk L., Marzecova A., Wolski P.
Institute of Psychology, Jagiellonian University, Kraków, Poland
Hemispatial neglect is a failure of attending to the left half-space resulting from lesions of the right hemisphere. It is believed to be an
attentional dysfunction, involving deficits in disengagement of attention from the objects in right visual field, as well as in inhibition of
return to the right visual field. Rosetti and Rode (1998) proposed a
method of ameliorating symptoms of neglect by means of prismatic
lenses, which shift vision (e.g. 15° to the right or left) and thereby
trigger changes in visuo-spatial integration. However, it is not clear,
which particular cognitive mechanisms are affected by the method.
Furthermore, leftward prism adaption in healthy subjects causes an
effect similar to hemispatial neglect. Similarly, cognitive and neural
mechanisms underlying this effect remain yet to be identified. Two
experiments were conducted, which aimed at indicating attentional
processes modified by prism adaptation in case of “neglect-like” effect in healthy individuals. Posner’s location-cuing task paradigm was
used in order to differentiate processes of endogenous and exogenous
orienting of attention, as well as to measure the effect of inhibition of
return. Participants performed the task before and after prism adaptation. Results are discussed in light of recent theories of neglect.
TIV.10
HYPERSENSITIVITY AND NOCICEPTIVE BEHAVIOUR
OF da-DREAM TRANSGENIC MICE
Krzyzanowska A.1, Benedet T.2, Barrio J.2, Naranjo J.2,
Avendano C.1
1
Department of Anatomy, Histology and Neuroscience, Autonomous
University of Madrid, Spain; 2 Department of Molacular and Cellular
Biology, Spanish National Center of Biotechnology, Madrid, Spain
DREAM is a Ca2-dependent transcriptional repressor that regulates
the expression of multiple genes, some of which are implicated in
pain mechanisms. To further investigate the role of DREAM in pain
we have developed transgenic lines of mice overexpressing dominant active mutants of DREAM (daDREAM). They were tested for
thermal and mechanical sensitivity in basal conditions and following
CFA-induced inflammation or CCI-induced neuropathic pain. Naïve transgenic (TG) mice showed significantly higher responses
to noxious heat as compared to wild type (WT). In a visceral pain
model,TG animals were markedly hyperalgesic in comparison to
WT. In models of chronic inflammation (CFA), TG animals show a
delay in the development of thermal hypersensitivity as compared to
the WT animals. In a model of neuropathic pain (CCI), within 5 days
post op, TG animals were as hyperalgesic as WT animals. However, considering that baseline sensitivity was lower in TG animals,
the degree of hyperalgesia was less than that of WT animals when
compared to pre-operative values. Thus, daDREAM is implicated in
various pain states as well as baseline noxious sensitivity.The behavioural data will be supplemented with histochemical data characterising sensory neurone populations in both groups of animals.
TIV.11
MODULATION OF P2X2/3 RECEPTORS BY OPIOIDS
IN RAT NODOSE NEURONES
Mamenko M.1, Chizhmakov I.1, Volkova T.1, Khasabova I.2,
Simone D.2, Krishtal O.1
1
Department of Cellular Membranology, Bogomoletz Institute of
Physiology, Kiev, Ukraine; 2 Department of Diagnostic and Biological
Sciences, University of Minnesota, Minnesota USA
P2X2/3 receptors, localized on peripheral and central terminals of
primary sensory neurons, are involved in persistent nociceptive signalling. Opioid peptides are known to produce analgesia through
central as well as via peripheral mechanisms. We have shown that
in rat nodose neurons P2X2/3-mediated responses were modulated by
opioids in a biphasic manner: an initial short phase of potentiation
(300–400 s) was followed by long-lasting inhibition of the response
(for about 50% at the steady-state level). Addition of GTP-gamma-S,
GDP to the intracellular solution and preincubation with pertussis
toxin indicated that P2X2/3 receptors were affected by opioids via Gprotein dependent pathways. We have also shown that sensitivity of
P2X2/3 receptors to endomorphin-1 is altered after co-culturing of nodose neurons with fibrosarcoma cells (NCTC 2472). In co-cultured
neurons ATP-activated currents with “slower” desensitization kinetics were less inhibited by the opioid. “Ultra-slow” responses were
completely insensitive to endomorphin-1. The occurrence of these
responses increased with the duration of co-culturing. Thus, coculturing decreases sensitivity of ATP responses to endomorphin-1,
which can account for low sensitivity of cancer pain to opioids.
TIV.12
THE EXTRASYNAPTIC AMPA RECEPTORS
FUNCTIONING IS ALTERED UNDER INFLAMMATORY
PAIN
Kopach O.1, Park J.2, Petralia R.3, Sotnik A.1, Belan P.1, Tao Y.2,
Voitenko N.1
1
Department of General Physiology of Nervous System,
Bogomoletz Institute of Physiology, Kiev, Ukraine; 2 Department of
Anesthesiology and Critical Care Medicine, Johns Hopkins University
School of Medicine, Baltimore, Maryland, USA; 3 Laboratory of
Neurochemistry, National Institutes of Health, Bethesda, Maryland,
USA
We have recently shown that synaptic Ca2+-impearmable AMPA receptors (AMPARs) internalization in dorsal horn neurons underlies
the maintenance of nociceptive hypersensitivity in inflammatory
pain. Here we have analyzed if trafficking of extrasynaptic AMPARs
is also changed during development and maintenance of persistent
pain. We report that Complete Freund’s Adjuvant (CFA)-induced
inflammation causes an increase in functional expression of extrasynaptic AMPARs in rat substantia gelatinosa (SG) neurons during
326
the maintenance rather than development of persistent pain. This
increase, revealed as a significant enhancement of AMPA-induced
membrane currents and [Ca2+]i transients, was observed only in neurons characterized by an intrinsic tonic firing properties whereas no
changes were observed in neurons exhibiting a strong adaptation. The
increase was also accompanied by an enhancement of surface GluR1
expression and of the total amount of cobalt-positive neurons indicating an increase in a pool of GluR2-lacking AMPARs in extrasynaptic
plasma membrane. These results suggest that functional changes in
extrasynaptic AMPARs of tonic SG neurons that are associated with
the maintenance of nociceptive hypersensitivity may also contribute
to inflammatory pain. We also suppose that there is a different contribution of tonic and transient neurons to the detection of peripheral
painful stimuli and to maintenance of nociceptive hypersensitivity.
TIV.13
ASSOCIATIVE LEARNING MODIFIES
REPRESENTATION OF VIBRISSAE IN SII CORTEX
–A 2DG STUDY
Debowska W., Siucinska E., Liguz-Lecznar M., Kossut M.
Secondary somatosenory cortex (SII) in rodents receives input
from vibrissae trough reciprocal cortico-cortical connections from
the barrel cortex and directly from thalamic sensory nuclei. In humans, SII is activated bilaterally during attentional tasks and it is
consider to play a role in tactile memory and sensimotor integration.
We examined it behavioral paradigms that modifies SI evokes also
plasticity in SII. We measured area of SII activation by stimulation
of a row of vibrissae previously activated in classical conditioning
paradigm, in which stimulation of a row of vibrissae was paired with
a tail shock. The training consisted of 3 daily 10 min sessions, during which 40 pairings were delivered. Activation was mapped with
[14C]-2-deoxyglucose (2DG) autoradiography one day after the end
of conditioning. The autoradiograms were analyzed with computerized image analysis system, which aligned the 2DG uptake pattern
with Nissl stain. We reported previously that conditioning results in
enlargement of cortical representation of the “trained” row of vibrissae in SI. Here we found that in SII the representation of the “trained”
row is increased bilaterally, by 37% on the average. The increase was
observed in cortical layers II/III and IV. Clearly, plasticity in SII is
not simply a reflection of changes in SI. It may be supposed that in
response to activation of a pathway involved in conditioning, structures involved in attention respond more strongly to sensory stimuli.
TIV.14
EXPLORATION OF NEW ENVIRONMENT REVEALS
EXTENSIVE DEPRIVATION-INDUCED PLASTICITY
Radwanska A., Filipkowski R., Zakrzewska R., Kossut M.
Sensory deprivation elicits alterations in the functional organization of
the primary somatosensory cortex. It was shown that plucking out all
but one row of whiskers in adult mice evokes broadening of the functional representation of the spared row, as measured with radioactive
2-deoxyglucose (2-DG) uptake during passive stimulation of whiskers. We would like to establish whether changes in metabolic activity
caused by sensory deprivation are paralleled by changes at genomic
levels visualized by immediate early genes immunohistochemistry.
Exploration of enriched environment is a powerful trigger to induce
immediate early genes in the barrel cortex. In this study we show that
plasticity of the functional representation of the spared row of whiskers
can be estimated by radioactive 2-DG method in animals actively using their whiskers while exploring new environment. Expansion of the
spared row representation in the deprived hemisphere reaches 140% of
the control (non-deprived) hemisphere after 1 week of deprivation and
spreads to whole barrel field after four weeks of deprivation. We also
show that induction of some early immediate genes during exploration
of new environment is limited to the non-deprived barrels after one
day of deprivation. In further course of studies we will perform immunohistochemical reactions for proteins encoded by early immediate
genes in brains from animals deprived for 1 or 4 weeks.
TIV.15
ESTIMATING FUNCTIONAL BRAIN CONNECTIVITY
THROUGH ANALYSIS OF MULTI-SITE EVOKED
POTENTIALS RECORDINGS
Sobolewski A., Kublik E., Swiejkowski D., Wrobel A.
Local field potential (LFP) – the result of summed postsynaptic potentials from cell populations – reveal the most characteristic neural
activity at the recording site in the brain. Thus LFPs are well suited
for study of neural networks, specifically those involved in processing
of sensory information. We propose a method of assessing functional
brain connectivity through LFP analysis. The method is applied to
multi-site signals representing potentials evoked by a repeated, stereotyped stimulus. In spite of a stereotyped stimulus, all responses
are different due to, inter alia, ongoing background activity of the
brain and this trial-to-trial variability is utilized in our analysis. The
method is based on calculation of correlations between trial-to-trial
LFP variations at every post-stimulus latency at every recoding site.
The results show how neuronal activities at different sites and latencies correspond to activation at other sites with a given time delays.
We used this method to analyze the functional connectivity in thalamocortical network involved in processing of somatosensory (vibrissal) information in non-anaesthetised rat. One result is that the
cortical activation at 25–50 ms post-stimulus correlates with thalamic LFP measured at 50–150 ms post stimulus, thus implying this
late latency thalamic activity depends on a corticothalamic feedback.
Apart from raw LFP, the method is applicable to results of various
decomposition methods of brain signals (PCA, ICA, etc.).
TIV.16
INTEGRATION OF MULTIPLE-WHISKER
STIMULATION IN RAT BARREL CORTEX
Kaminski J., Kublik E., Leski S., Wrobel A.
The vibrissal system of rodents has become one of the dominant
models for investigating the mechanisms of sensory information
processing. However, the mechanisms underlying integration of
multiple whisker input is not well understood. To address this question, we recorded local field potentials (LFP) from the barrel cortex
in of anaesthetised rat. Recording points were distributed on the 4
× 22 grid covering two neighbouring cortical columns and septa
between them. Potentials evoked (EP) by deflection of single whiskers and sets of three whiskers (within arc and/or row) were analy-
sed by two dimensional current source density method (2D CSD).
The multiple whisker response was compared to linear predictor,
defined as a sum of corresponding single whiskers responses. CSD
performed on the data revealed significant differences between
linear prediction and multiple whisker deflection. Multiple input
responses had lower amplitudes as compared to linear prediction
condition. The earliest differences were observed in infra- and
supragranular layer approximately 8 ms after stimulation. Differences in granular layer appeared 10 ms after stimulation. Our data
suggest that supra- and infra granular layers are involved in initial
phase of the integration of multiple whisker inputs
TIV.17
SPINAL CORD SORTILIN DISTRIBUTION AND
LEVELS CHANGE IN REGION-SPECIFIC MODE AFTER
SPINALIZATION: p75 COMPLEXING
IN MOTONEURONS?
Gajewska O.1, Mankovskaya T.1, Ziemlinska E.1, Korczynski J.2,
Czarkowska-Bauch J.1, Skup M.1
1
Department of Neurophysiology, 2 Laboratory of Confocal
Microscopy, Nencki Institute of Experimental Biology PAS,
Warszawa, Poland
Membrane receptor sortilin is involved in sorting and processing of
proteins. In complex with p75 receptor binds proneurotrophins what
can lead to apoptosis and remodeling of neuronal network. To study
sortilin involvement in spinal cord (SC) remodeling after injury, we
characterized its distribution patterns, levels and relation to p75 expression in L3/L4 segments, 6 weeks after SC transection at low thoracic level. In intact rats sortilin immunoreactivity (IR) was widely
distributed in white and grey matter. The strongest IR was observed
in glial cells of lateral and ventral funiculi. In the grey matter sortilin IR appeared in number of neurons and glial cells, except for the
lamina 2, where it rarely occurred. In contrast, p75 was limited to the
bundle of strongly IR fibers in dorso-lateral lamina 2. Sortilin and
p75 did not colocalise there and overlapped in isolated large neurons
of lamina 9. Spinalization caused a 5% decrease of sortilin IR in the
white matter not accompanied by p75 IR changes. In the grey matter sortilin IR level was not changed but frequency of p75/sortilin
IR overlapping increased in lamina 9 neurons. This result indicates
postlesion increase of p75/sortilin complexing in motoneurons which
may reflect activation of proneurotrophin-mediated dysfunction.
Our data show lack of such interaction in primary sensory afferents.
Sortilin IR in numerous cells devoid of p75 labeling confirms that it
plays also other roles. Supported by MSE P-N/029/2006 grant.
TIV.18
DIFFERENCES IN MORPHOMETRIC PROPERTIES OF
MUSCLE FIBERS AND INNERVATION RATIO OF MALE
AND FEMALE MOTOR UNITS IN THE RAT MEDIAL
GASTROCNEMIUS MUSCLE
Mierzejewska-Krzyzowska B.1, Drzymala-Celichowska H.2,
Celichowski J.2, Bukowska D.2
1
Department of Anatomy in Gorzow, 2 Department of Neurobiology,
University School of Physical Education in Poznan, Poznań, Poland
The study aimed at demonstration of sexual differences in morphology and innervation of the medial gastrocnemius muscle (MG) in
male and female Wistar rats. The experimental project involved three
stages: (1) the functional isolation and analysis of contractile properties of as many motor units (MUs) as possible, (2) recording the
contraction force of the whole muscle evoked by stimulation of the
sciatic nerve and, (3) the morphological examinations of MG. The
experiments revealed that MG of males contained 46% of fast fatigable (FF) MUs, 40% of fast resistant (FR) MUs and 14% of slow (S)
MUs, whereas in females 40% of FF, 37% of FR and 23% of S type
MUs were found. The force of MUs in male muscle was higher, and
the twitch time parameters were longer in males. Moreover, the muscle mass and force in males was significantly higher. The relation of
the muscle force to the mean value of MUs force revealed that male
MG contained approximately 10% more MUs than in females. The
male muscles which had approximately 1.5 times bigger mass than
in females were composed of over 11 800 muscle fibers, whereas in
females 8 000 fibers. These results strongly suggested that the average number of muscle fibers in individual MUs of males was higher
about 47% comparing to females. In addition, the mean diameter of
muscle fibers was 29% bigger in males. Finally, the cross-section
area of muscle fibers was also 15% larger in males.
TIV.19
SUMMATION OF MOTOR UNIT FORCES IN THE RAT
MEDIAL GASTROCNEMIUS MUSCLE
Drzymala-Celichowska H., Krutki P., Celichowski J.
Department of Neurobiology, University School of Physical
Education, Poznań, Poland
The purpose of the study was to examine the summation of forces of
two and four individual motor units (MU), and four groups of units in
the medial gastrocnemius muscle during parallel stimulation of axons
innervating these units. Experiments were performed on Wistar rats
under pentobarbital anesthesia. Single motor axons were prepared by
splitting the L5 and L4 ventral roots until electrical stimulation of
these filaments evoked “all-or-none” type motor unit action potentials
and twitch forces in the studied muscle. The examination of groups
of units followed division of the L5 ventral root into four bundles of
axons. After the isolation, a number of activities were recorded: single
twitches, unfused and maximal tetani. The effects of summation of
motor unit contractions were evaluated as differences between the
recorded force during parallel stimulation and the algebraic sum of
individual forces. As more units were activated simultaneously it was
found that the recorded force became progressively lower than the estimated algebraic sum of forces. Furthermore, in all cases, the force of
unfused tetani summated more effectively than the force of twitches
or of fused tetani. Finally, a decrease in the amplitude of force oscillation in the unfused tetanus was observed in relation to values expected based on algebraic summation of oscillations for individual
MUs. The main finding of this study is that forces produced by MUs
during simultaneous stimulation summate nonlinearly.
TIV.20
INFLUENCE OF 5-WEEK WHOLE BODY VIBRATION
TRAINING ON MOTOR UNIT CONTRACTILE
PROPERTIES IN THE RAT MEDIAL GASTROCNEMIUS
MUSCLE
Lochynski D., Celichowski J., Kaczmarek D., Krutki P.
The aim of the study was to determine the effect of the whole body
vibration training on motor unit contractile properties in rat medial
gastrocnemius.Two groups of Wistar rats trained 30 s (V30, n=4) or 4
× 30 s (V120, n=4) daily, 5 days weekly, for 5 weeks on vibratory plat-
328
form (Power Plate®, USA, 2 mm vibration at 50 Hz) were compared
to the control group (C, n=10). The employed vibration excited motor
units through the stretch reflex loop. Functional isolation of units was
achieved by electrical stimulation of thin filaments of the ventral roots.
A total of 267 motor units were studied (56 in V30, 69 in V120, and 142
in C). They were classified into: fast fatigable (FF), fast resistant to fatigue (FR) and slow (S) types.In the V30 and V120 group, no changes
in the distribution of motor unit types were noted. For both groups the
specific and the only change was a significant increase in the twitch
and maximal tetanus force in FF motor units. Additionally, for V120
group an increase in maximal tetanus force of S units was noted. For
V120 group the contraction and relaxation times shortened in fast motor units and FF motor units became also less resistant to fatigue. It is
shown that only a little increased daily physical activity induced by
vibration considerably influences motor unit properties and FF motor
units seem to be the most sensitive to vibratory stimulus.
TIV.21
AGEING AFFECTS THE RATE OF FORCE
DEVELOPMENT OF MOTOR UNITS IN RAT FAST
MUSCLE
Kaczmarek D., Lochynski D., Celichowski J., Krutki P.
The rate of force development (RFD) of skeletal muscle decreases
with ageing. The purpose of this study was to determine whether this
phenomenon may be connected with the age-related changes in motor unit (MU) properties. One group of young (5–10 months old, n=9)
and two groups of old (24–25 months old, n=6 and 28–30 months
old, n=8) male Wistar rats were studied. Contractile activity of isolated MUs in the rat medial gastrocnemius muscle was evoked by
electrical stimulation of ventral root filaments. MUs were classified
into fast fatigable (FF), fast resistant (FR), and slow (S) according
to susceptibility to fatigue and twitch contraction time. The RFD,
force and force-time integral (FTI) of twitch, contraction following
doublet pulse stimulation and maximal tetanus were measured and
compared. Absolute and relative RFD of studied MUs evidently increased with ageing. We observed gradual augmentation of absolute
force and FTI of evoked doublet contractions during ageing in S MUs
while in FF units this increase was transient (only for 24–25 months
old animals). However, the relative values of force and FTI after doublet stimulus were lower in S and FR MUs and unchanged in FF units
in old animals as compared to the young. The increase in MU RFD is
opposite to decrease in muscle RFD with ageing. The decline of the
muscle performance capabilities can not be attributed to deterioration of mechanical parameters of survived MUs but rather is due to
overall loss and change in proportion of MUs.
TIV.22
VARIABILITY OF CONTRACTILE PROPERTIES AND
ACTION POTENTIALS OF MOTOR UNIT IN FLEXOR
DIGITORUM BREVIS AND MEDIAL GASTROCNEMIUS
MUSCLES IN THE RAT
Ciechanowicz-Kowalczyk I., Celichowski J.
The differences between motor unit (MU) properties in foot (flexor digitorum brevis, FDB) and sural (medial gastrocnemius, MG)
muscles in anaesthetized Wistar rats were studied. In experiments
functional isolation of single MUs was achieved by stimulation of
thin filaments from the L5–L6 and L4–L5 ventral roots for FDB
and MG muscles, respectively. The mean muscle mass amounted
to 76 mg for FDB and 895 mg for MG. The force and the motor
unit action potential (MUAP) properties were analysed. The three
types of MUs (FF, FR and S) in the MG muscle were found. Sag
phenomenon was visible in all fast units at 40 Hz stimulation. In
FDB, the slow MUs were not noted. Sag was visible for only a part
of fast MUs at lower frequencies of stimulation. In the MG muscle
the fatigue index had bimodal distribution, whereas in FDB the
distribution was continuous. The twitch and maximum forces
were approximately 8 times lower in FBD then in MG, whereas
the contraction time was significantly longer. The force-frequency
curves of all motor units in FDB were shifted towards lower frequencies and comparison to the same type of MUs in MG. The
MUAP durations for FF and FR MUs were longer whereas the
MUAP amplitudes were higher for FF units in MG in relation to
FDB. In conclusion, the variability of MU properties in distal and
proximal muscle concerns all studied contractile parameters and
likely reflects different motor control strategy.
TIV.23
DOUBLET OF ACTION POTENTIALS EVOKED BY
INTRACELLULAR INJECTION OF CURRENT INTO
RAT MOTONEURONES
Mrowczynski W., Krutki P., Celichowski J.
Experiments were based on electrophysiological method of direct
stimulation and recording from motoneurones located in L4–L5
segments of the rats spinal cord. Records from spontaneously firing motoneurones were collected after intracellular application
of a current of gradually increasing intensity. Current threshold
for generation of the first action potential was determined. Additionally, intervals between six first potentials were analyzed.
It was demonstrated that the gradually increasing current intensity caused the generation of doublets in 68% of motoneurones
studied. Doublets appeared when stimulating current increased to
120–500% of the threshold value. Moreover, the appearance of the
doublet resulted in a significant prolongation of time interval to
the subsequent, third action potential. These results suggest that
doublets in motoneuronal firing appear as a result of their strong
synaptic excitation. The prolonged interpulse interval after the
doublet firing can be due to an increase of duration and amplitude
of afterhyperpolarization (AHP), previously observed in antidromic doublet stimulation.
TIV.24
CHANGES IN THE FORCE-FREQUENCY
RELATIONSHIP AFTER THE TREADMILL AND
WHOLE BODY VIBRATION TRAINING OF MOTOR
UNITS IN RAT MEDIAL GASTROCNEMIUS MUSCLE
Baczyk M., Lochynski D., Celichowski J.
The aim of the study was to compare changes in the force-frequency
relationship of motor units (MUs) in rat medial gastrocnemius muscle, caused by treadmill training and whole body vibration training.
24 Wistar rats were studied. Rats were divided into 3 groups: tread-
mill training group (n=7, locomotion speed 27 cm/s, 1 km daily, 5
days weekly, for 4 weeks), whole body vibration training group (n=6,
Power Plate® vibration platform, 50 Hz vibration of 2 mm amplitude,
30 s daily, 5 days weekly, for 4 weeks) and a control group n=11.
Functional isolation of single MUs was achieved by electrical stimulation of ventral roots filaments. A total of 424 MUs were studied (148
in treadmill training, 142 in vibration training and 134 in control).
MUs were classified as fast fatigable – FF, fast resistant – FR or S –
slow. The frequency causing 60% of maximum force of MUs has decreased in FF units of both trained groups and increased in FR units.
In S units the increase of the parameter was noted only in treadmill
trained group. Additionally, the slope of the force-frequency curve
has increased in all MUs types in the vibration training group.
TIV.25
CHANGES IN MOTOR UNITS PROPERTIES IN A RAT
MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
(ALS)
Krysciak K., Celichowski J.
Department of Neurobiology, Univerity School of Physical Education
ALS is a fatal neurodegenerative disorder characterized by developing
muscle weakness and paralysis which is an effect of selective and progressive death of motoneurons. The experiments were carried out on
motor units (MUs) of the medial gastrocnemius muscle in transgenic
rats with hSOD1(G93A) mutation. The animals were divided into three
groups: 3 months old, 4 months old and approximately 200 days old
in nearly terminal state, when nerve stimulation revealed on average
6 MUs in the studied muscle. Because of considerable disturbances
in contraction time (CT) and disappearance of sag effect, usually applied for MUs classification as fast and slow, the division of MUs was
based on 20 Hz tetanus index. Meanwhile, to distinguish fast fatigable
(FF) from the fast resistant (FR) MUs the fatigue index (FatI) was applied. Results showed that MUs proportion changed radically in the
course of the disease and in terminal state an increase of S and a decrease of fast MUs was observed. Moreover, CT increased gradually
in all MU types. FatI of FF MUs increased but, at the same time, for
FR and S gradually decreased. Twitch force as well as tetanus force in
terminal group increased especially for FR and S MUs what indicates
plastic changes in examined muscle (sprouting of surviving axons and
collateral reinnervation), leading also to increased amplitude of MUs
action potentials. EMG analysis additionally showed slowing of axonal conduction velocity or/and motor plate transduction.
TIV.26
SUMMATION OF MMG SIGNALS DURING
CO-ACTIVITY OF TWO ISOLATED MOTOR UNITS
IN MEDIAL GASTROCNEMIUS MUSCLE OF THE RAT
Kaczmarek P.1, Celichowski J.2, Drzymala-Celichowska H.2,
Kasinski A.1
1
Institute of Control and Information Engeenering, Poznan University
of Technology, Poznań, Poland; 2 Department of Neurobiology,
The MMG signal generated in contracting pennate muscle is due to a
transversal displacement of the its surface. It was shown on the base
of in vivo experiments and a computer model that the MMG signal
recorded during an isolated motor unit (MU) contraction is dependent
on the stimulation frequency, the position of the laser distance sensor
(LDS) and MU architecture. The three different profiles of the MMG
can be observed. The first type denoted as N (negative) is observed for
the LDS located over the proximal connection of the MU (the distance
from the muscle surface decreases with contraction force increase). In
class P (positive) the relationship is opposite to N and the LDS is located over the area between distal connection of the MU and tendon of
insertion. Finally, for the third class denoted as M (mixed) the MMG is
initially positive, and when the contraction force exceed a certain level
it starts to decrease and becomes negative. The process of the MMG
summation during two MUs contraction was also investigated. It was
observed that for the MUs with MMG-N or MMG-M profile the MMG
summation was quasi linear. In case of twitch and unfused contractions it was equal the algebraic sum of the individual MMGs. In case
of the fused contraction the resulted MMG was slightly lesser than
the algebraic sum. In opposition, the contraction of two MUs MMG-P
induced the MMG signal lesser than the algebraic sum and the resulted
signal was nearly equal the MMG presented by stronger MU.
TIV.27
CONTRACTILE PROPERTIES AND DISTRIBUTION
OF TYPES OF MOTOR UNITS INNERVATED BY AXONS
FROM L4 AND L5 VENTRAL ROOTS IN THE RAT
MEDIAL GASTROCNEMIUS
Taborowska M., Celichowski J.
There is a limited data concerning the rostro-caudal distribution of
motoneurons within the motor nucleus. In present experiments single motor units in rat medial gastrocnemius were studied and their
contractile properties as well as percentage participation of different
types of motor units belonging to subpopulations innervated by axons
in L4 and L5 ventral roots were analyzed. Experiments were based on
functional isolation and electrical stimulation of as many as possible
axons from L4 and L5 ventral roots. 35 motor units innervated by L4
and 90 units innervated by L5 ventral root were studied. We found
that the composition of the three physiological types of motor units
(fast fatigable, fast resistant and slow) in the two subpopulations of
motor units was similar. However, the twitch time parameters (the
contraction time and half-relaxation time) were slightly longer in L4
in comparison with L5 subpopulation (the difference was significant
for fast resistant units) although the difference has not been reflected in
expected shift of a steep part of force-frequency of stimulation curve
towards lower frequencies in L4 subpopulation of motor units. Force
parameters were similar for motor units belonging to two subpopulations. The small differences between L4 and L5 subpopulations of
motor units can be due to possible differences in a spatial distribution
of their muscle fibres in the semipennate medial gastrocnemius.
TIV.28
THE TETANIC DEPRESSION IN FAST MOTOR UNITS
OF MAMMALIAN SKELETAL MUSCLE CAN BE
EVOKED EVEN BY LENGTHENING OF ONE INITIAL
INTERPULSE INTERVAL
Sowinska Z., Celichowski J., Krutki P., Lochynski D.
The tetanic depression is phenomenon observed when higher frequency of stimulation of fast motor units immediately follows the lower
one. However, it is not known does only one prolonged first interpulse
interval can evoke this effect. This study was conducted on 27 fast
330
motor units of cat and 47 units of rat medial gastrocnemius muscles. In
experiments performed on the two species the following stimulation
protocol was applied in the main part of experiment: (1) the 6-pulses
train of stimuli at 20 Hz (cat) or 40 Hz (rat), i.e. at 50 and 25 ms interpulse intervals, (2) the 6-pulses train of stimuli with the first interpulse
interval prolonged from 50 ms to 100 ms (cat) or from 25 to 50 ms (rat)
followed by 5 stimuli at 20 Hz (cat) or 40 Hz (rat), (3) the 6-pulses train
of stimuli at 20 Hz (cat) or 40 Hz (rat). Effects of two-time prolongation of the first interpulse interval were observed as a decrease of the
force following the sixth stimulus in a train. The tetanic depression in
rat motor unit tetani for fast fatigable (FF) units amounted to 5.39 ±
4.94% and 9.24 ± 4.13% for fast resistant (FR) units whereas in cats the
mean values were 15.00 ± 10.15% and 27.00 ± 13.95% for FF and FR
motor units, respectively. This results prove that the studied phenomenon influence the force development when the motoneuron begins the
activity even with only one, first prolonged interpulse interval.
TIV.29
THE LINEAR SUMMATION OF THE MOTOR UNITS
ACTION POTENTIALS RECORDED WITH WIRE
ELECTRODE IN THE RAT MEDIAL GASTROCNEMIUS
MUSCLE
Mazurkiewicz P.1, Kasinski A.1, Drzymala-Celichowska H.2,
Celichowski J.2
1
Institute of Control and Information Engeenering, Poznan University
of Technology, Poznań, Poland; 2 Department of Neurobiology,
University School of Physical Education in Poznań, Poznań, Poland
The summation of the motor units action potentials (MUAPs) in
the rat medial gastrocnemius muscle was studied. Experiments
were performed on anesthetized animals and single motor units
(MUs) were functionally isolated by electrical stimulation of thin
filaments of ventral roots. MUAPs were recorded with two silver
wires inserted into the muscle. Algebraic sum of the MUAPs recorded from two to four individual MUs were compared to the
action potentials recorded during their simultaneous stimulation.
The peak-to-peak amplitude, the longitudinal center of the signal
square, the number of phases and turns, correlation coefficient
and mean-square error were measured. In all studied cases of
summation the number of phases and turns were the same in the
two compared signals. When summation effects of two MUs were
compared, there were no significant differences in the peak-topeak amplitude. When 3 and 4 MUs were co-activated significant
differences between a sum of individual MUAPs and the recorded action potential were noted in some cases. The most variable
parameter was the longitudinal center of the signal square which
was the major source of differences. It was also shown that in a
case of contractions of low force MUs the studied parameters of
the algebraic sum of their MUAPs was similar to parameters of
action potentials recorded during simultaneous stimulation.
TIV.30
PROJECTIONS FROM THE BASILIAR PONTINE
NUCLEI TO THE CEREBELLAR CORTEX:
FLUORESCENT TRACING STUDY IN THE RABBIT
Zguczynski L.1, Bukowska D.2, Mierzejewska-Krzyzowska B.1
1
Department of Anatomy in Gorzow, 2 Department of Neurobiology, University School of Physical Education in Poznan, Poznań,
Poland
Pontine nuclei (PN) are large center transferring cortical afferents to the cerebellum. The study addresses to distribution
of PN neurons in the pontocerebellar link. Two different neuroplasmatic and nuclear fluorescent tracers were injected in
separate experiments into: (1) paramedian lobule (PML) and
pyramis, and (2) pyramis and uvula. Following both combination of injections, retrogradely labeled neurons were found
bilaterally in all PN apart from the ventral nucleus. These neurons, parent for the pontocerebellar projection, clustered in two
groups. First larger group occupied the dorsolateral nucleus and
the dorsolateral region of lateral and peduncular nuclei. Second
smaller group involved the dorsal region of paramedian nucleus
and the dorsomedial region of peduncular nucleus. Projection to
the uvula arises from nearly entire rostrocaudal extent of PN,
and that to the pyramis and PML originates from medial and
caudal extent of PN. In spite of substantial overlap of projection area in PN, no neurons were found to project by way of
axonal collaterals. The strongest PN projections arise from the
dorsolateral nucleus and these from the paramedian, lateral and
peduncular nuclei are about two, three and five times weaker,
respectively. PML and the uvula receive the most numerous
projections (mean 4 744 and 4 283 neurons) whereas projection
to the pyramis (mean 1 172 neurons) is apparently smaller. The
differences in the projection seem to be cause of different function of these lobules.
TIV.31
PROJECTION FROM THE DORSAL COLUMN
NUCLEI TO THE CEREBELLAR CAUDAL VERMIS:
COMPARISON OF COLLATERAL AND NONCOLLATERAL CONNECTIONS
Bukowska D.1, Zguczynski L.2, Mierzejewska-Krzyzowska B.2
1
Department of Neurobiology, 2 Department of Anatomy in Gorzow,
The study was performed to determine projection from the
dorsal column nuclei (DCN), both independent and by way of
axonal collaterals, to the pyramis and uvula. Two different fluorescent tracers were unilaterally injected into the two vermal
targets. Single and double retrogradely labeled neurons, parent
for non-collateral and collateral projections, were found in defi ned regions of DCN. Non-collateral bilateral projections directed to the pyramis and uvula are stronger to the latter, and
originate from neurons in the lateral cuneate nucleus (CuL; n=
5 040 vs. 6 637), the complex of gracile and medial cuneate nucleus (Gr+CuM; n=820 vs. 2 190) and CuM (n=32 vs. 92). Weak
projection from the dorsal Gr (n=57) is destined for the pyramis. Neurons in the rostral and middle CuL, projecting to the
pyramis and uvula occupy ventrolateral and ventromedial positions, respectively, and a narrow region between them is common. More caudally, small areas in CuL, Gr+CuM and CuM
supply the uvula, but in majority, areas of projection from these
subnuclei to both lobules overlap. Weaker ipsilateral projections
by way of axonal collaterals (n=175) arise from neurons in common projection areas in CuL and Gr+CuM. To sum up, neurons
for the DCN-pyramis and DCN-uvula projections vary in number and distribution, and there is a small population of neurons
participating in divergent projection to both lobules. Different
extent of DCN influences may be due to disparate function of
the pyramis and uvula.
Poster Session II 331
TIV.32
DISTRIBUTION OF THE PARVALBUMIN, CALBINDIND28K AND CALRETININ IMMUNOREACTIVITY IN
THE STRIATUM OF THE BRAZILIAN SHORT-TAILED
OPOSSUM
Majak K.1, Domaradzka-Pytel B.1, Spodnik J.1, Turlejski K.2,
Djavadian R.2, Morys J.1
1
Department of Anatomy and Neurobiology, Medical University of
Gdansk, Gdańsk, Poland; 2 Department of Neurophysiology, Nencki
To understand the organization of inhibitory circuitries in the
striatum of the opossum, the distribution of parvalbumin (PV),
calretinin (CR) and calbindin (CB) was investigated within the
nucleus accumbens (Acc), caudate nucleus (Cd) and putamen (Pu).
Brains from six adult opossum (both sexes) were stained for PV,
CR and CB and analyzed using fluorescent or confocal microscopy. Within neurons immunostained for each calcium-binding
protein four major types of neurons were distinguished. Type 1
– small ovoid or roundish somata with three to five thin dendrites
of approximately equal thickness; type 2 – medium-to-large-sized
multipolar neurons with dendrites of variable thickness; type 3 –
fusiform neurons of variable size emanating dendrites from the
opposite poles of the somata; type 4 – pyramidal neurons. Moderate-to-high density of CB immunoreactive (-ir) neurons was
observed in the Cd and Pu, but staining in the Acc was lighter.
Light density of PV-ir neurons was present in the Cd, Pu and Acc,
but PV-immunoreactivity of neuropil was high. Only single CR-ir
neurons were scattered through the all studied structures, but the
immunostaning of neuropil was much higher. Our data provide
baseline information for comparisons of distribution of calcium
binding proteins in different species, including rat, monkey and
human.
TIV.33
CHANGES IN EXPRESSION OF nNOS AND
PARVALBUMIN IN DORSAL HORN OF THE LUMBAR
SPINAL CORD AFTER SPINAL CORD INJURY
Davidova A., Capkova L., Schreiberova A., Lukacova N.
Department of Signaling Molecule, Institute of Neurobiology Slovak
Academy of Sciences, Kosice, Slovak Republic
The aim of the present study was to examine changes in the level
of neuronal nitric oxide synthase (nNOS) and parvalbumin (PV)
in dorsal horn of the rat lumbar spinal cord segments (L2–L6) 7
and 14 days after thoracic spinal cord transection. Our data show
that staining of control transverse spinal cord sections with nNOS
antibody provided an intense labeling of small round or bipolar
nNOS- immunoreactive (IR) cells bodies, fibers and terminal
like structures which were distributed throughout lamina II and
formed a dense IR band. The large multipolar nNOS-IR cells
with stellate or elongated cell bodies were occasionally seen in
the deeper layers (laminae III–IV). A decreased density of fiberlike and the loss of somatic nNOS staining was found in dorsal
horn layers after transection at both time points. A quantitative
assessment of nNOS IR disclosed a strong decrease in the number
of small neurons in superficial laminae (laminae I–III). In addition, an intense PV-IR band, consisting of terminal like structures
in lamina II under normal conditions, was significantly reduced
due to spinal cord transection. The present study indicates a considerable decrease in the number of nNOS-IR neurons in super-
ficial dorsal horn, where NO modulates the activity of inhibitory
GABA-ergic interneurons. The changes in expression of both the
calcium binding proteins in dorsal horn may modulate nociceptive transmission. Support: APVV grant 0314-06 and VEGA Grant
2/0015/08 from the SAS.
POSTER SESSION II
TV: Neural Excitability, Synapses and Glia:
Synaptic Plasticity and Networks
TV.01
CENTRAL RETINAL LESIONS INDUCE TIMEDEPENDENT PROTEIN EXPRESSION CHANGES
THROUGHOUT AREA 17
Hu T.1, Van den Bergh G.1, Eysel U.2, Heylen K.1, Arckens L.1
1
Laboratory of Neuroplasticity and Neuroproteomics, K.U. Leuven,
Lueven, The Netherlands; 2 Department of Neurophysiology, Ruhr
University Bochum, Bochum, Germany
Central retinal lesions lead to loss of visual input in the central
part of area 17 of the adult cat. Eventually this results in topographic map reorganization within the sensory-deprived cortical
lesion projection zone (LPZ) as measured by electrophysiology.
This recovery coincides with changes in gene/protein expression. We therefore investigated these alterations as a function of
post-lesion survival time in three regions of primary visual area
17: the center of the LPZ, the border of the LPZ and far peripheral
area 17. We analysed the differential protein expression patterns
between control and experimental animals with 2-dimensional
differential gel electrophoresis. We have traced 74 differential
spots for the three regions in area 17. Central retinal lesions
clearly induced region-specific and time-dependent protein expression changes. Even the protein expression in far peripheral
area 17 spared from a direct impact of the central retinal lesions
differed from the corresponding region in normal subjects. We
succeeded in identifying 93% of the differential spots by means
of mass spectrometry and software processing helped functional
grouping within the list of proteins. This analysis allowed us to
select two plasticity-related proteins for further validation experiments.
TV.02
A SINGLE EXPOSURE TO AN ENRICHED
ENVIRONMENT STIMULATES THE ACTIVATION
OF DISCRETE NEURONAL POPULATIONS
IN THE BRAIN OF THE fos-tau-lacZ MOUSE
Ali A., Wilson Y., Murphy M.
Department of Anatomy and Cell Biology, The University
of Melbourne, Melbourne, Australia
Storage of experience, including learning and memory, is thought
to involve plasticity within pre-existing brain circuits. One model for looking at experience-dependent changes is environmental
enrichment (EE), which involves exposing animals to a complex
novel environment. Animals exposed to EE have previously
been shown to exhibit a variety of behavioural and structural
alterations in the brain, including decreased stress, improved
332
learning and memory, altered levels of immediate early genes
and synaptic change in the visual cortex. We were interested in
understanding what regions of the brain are activated during the
initial stages of EE. We used the fos-tau-lacZ (FTL) transgenic
mouse to examine changes in functional activation throughout
the brain after a single exposure to EE. Female C57BL6 FTL
mice (n=19) were divided into three groups; enriched, home cage
and handled control. We found that early in the process of EE,
there was a high level of FTL expression in a series of brain
regions in the enriched group compared to the home cage group
and the handled control group, indicating that multiple circuits
were activated. These regions include the claustrum, infralimbic
cortex, hippocampus, amygdala and the hypothalamus. We believe that EE stimulates an initial strong increase in activation
of multiple functional circuits. These circuits are presumably
involved in the initial response of the animal to the enriched
environment.
TV.03
DOPAMINERGIC D1/D5-RECEPTOR BLOCKADE
WITHIN THE NUCLEUS ACCUMBENS AND
ITS EFFECT ON HIPPOCAMPAL LONG-TERM
POTENTIATION IN THE DENTATE GYRUS OF FREELY
MOOVING ANIMALS
Tabassum H., Frey J.
Magdeburg, Germany
Hippocampal long-term potentiation (LTP) is considered to be a
cellular model of learning and memory. It has been shown that
stimulation of modulatory brain regions can influence hippocampal LTP. Quite little is known of the role played by the nucleus
accumbens (NAc) on hippocampal LTP. The NAc is the central
component of the basal ganglia positioned to integrate signals
arising from corticolimbic areas and to translate them into motor
behavior. Electrical stimulation of the NAc core and shell region
has been shown to modulate LTP in the dentate gyrus. We were
now interested to study if the dopaminergic system within the
NAc affects LTP in the dentate gyrus in freely moving animals.
Thus, we have applied a specific dopaminergic D1/D5-receptor
blocker, SCH23390, into the NAc and studied its possible effect
on control potentials as well as on LTP in the dentate gyrus in
freely moving rats. Interstingly, treated animals show a depression in baseline responses recorded in the dentate gyrus, just by
D1/D5-blockade within the NAc. Furthermore, these animals
were characterized by a reduced LTP after weak tetanic stimulation of the perforant path when compared with controls. As there
is no direct dopaminergic connection between NAc and dentate
gyrus, the effects must involve indirect mechanisms, such as glutamatergic and/or GABAergic pathways.
TV.04
DIFFERENT REGIONS OF THE APICAL BRANCH OF
CA1 DENDRITES EXPRESS SPECIFIC FORMS OF LTD
IN HIPPOCAMPAL SLICES IN VITRO
Parvez S., Ramachandran B., Frey J.
Magdeburg, Germany
Long-term potentiation (LTP) and long-term depression (LTD)
of excitatory synaptic transmission are widespread phenom-
ena expressed at many excitatory synapses in the mammalian
brain. Because of its long duration, input specificity and associative properties, LTP and LTD have been used as cellular
models for memory. We wanted to investigate whether different locations on the apical dendritic branch could influence the
induction of LTD and its related properties. Late-LTD could be
induced in the apical CA1 dendrites by strong low-frequency
stimulation (SLFS) pattern if the synapses were located distally, whereas, proximally located synapses were not able to
maintain late-LTD. However, SLFS in both locations was able
to trigger the synthesis of plasticity-related proteins, which
could be evidenced by cross tagging experiments. In addition,
we have investigated if hippocampal CA1-LTP prevents/occludes the establishment of LTD in the same synaptic input at
specific time points after LTP-induction. We show induction of
LTP occludes longer-lasting but not short-term LTD about 1 h
after LTP-induction. However, after 4 h, i.e. after transformation of early- into late-LTP, also later forms of LTD can again
be induced in the same synaptic input. Our results demonstrate
that hippocampal neurons do not lose their capacity for the
induction of longer forms of LTP or LTD after the establishment of late-LTP in the apical dendrites of hippocampal CA1neurons.
TV.05
THE EFFECTS OF PROTEIN SYNTHESIS INHIBITOR
ON THE EXPRESSION AND RE-CONSOLIDATION
OF PENTYLENETETRAZOL KINDLED SEIZURES
Maciejak P.1,2, Szyndler J.2, Turzynska D.1, Sobolewska A.1,
Taracha E.1, Skorzewska A.1, Lehner M.1, Krzascik P.2,
Hamed A.2, Bidzinski A.1, Plaznik A.1,2
1
Department of Neurochemistry, Institute of Psychiatry and
Clinical Pharmacology, Medical University of Warsaw, Warszawa,
Poland
In the present study the effects of a protein synthesis inhibitor, cycloheximide (125 μg, i.c.v.), on the expression and reconsolidation of pentylenetetrazol-induced kindled seizures,
were studied in rats. Cycloheximide given repeatedly (every
second day) to fully kindled rats, immediately after 4 consecutive sessions of PTZ-seizures, did not modify the strength of
subsequent fits of convulsions. On the other hand, the protein
synthesis inhibitor significantly attenuated the strength of convulsions when the drug was administered 1 h before the PTZ
injection, every second day for 5 consecutive experimental
sessions. However, when cycloheximide was omitted in a consecutive session, PTZ induced a fully developed fit of tonicclonic convulsions, indicating that cycloheximide-induced
changes in seizure intensity were transitory, not related to a
stable modification in the function of neuronal circuits responsible for kindling seizures. The present fi ndings suggest that
the mechanisms underlying epileptogenesis are very resistant
to modification, and as such, are not the subject to permanent
changes even under the influence of protein synthesis inhibition. One possible reason may be the depth and multiplicity of
changes induced by seizures (i.e. alterations in enzymes, receptors, structural proteins, growth factors, etc.), that may cause
permanent biochemical and morphological alterations in the
brain that give rise to the kindled seizures.
TV.06
REINFORCEMENT OF AN EARLY-LTP IN THE
HIPPOCAMPAL CA1 BY STIMULATION OF THE
VENTRAL TEGMENTAL AREA IN FREELY MOVING
RATS
Scherf T., Frey J., Frey S.
Department of Neurophysiology, Leibniz-Institute for Neurobiology,
Magdeburg, Germany
It has been shown that the prolonged maintenance of hippocampal long-term potentiation (LTP) requires heterosynaptic
events during its induction. The ventral tegmental area (VTA),
a modulatory input to the CA1 region is a heterogeneous group
of dopaminergic cells and a major component of the mesolimbic
dopamine system. We used a method which allowed us to simultaneously record both, the field-EPSP and the population spike
(POP) in the hippocampal CA1 in freely moving rats by stimulation of the contralateral CA3. We could show that an early-LTP
in the CA1 region can be reinforced into a longer-lasting form by
high-frequency stimulation of the VTA 15 min after its induction. This reinforcement of an early-LTP in CA1 was dependent
on dopaminergic receptor activation and was also dependent on
protein synthesis. We have now extended our studies and could
show that high-frequency stimulation of the VTA alone, i.e. without LTP-induction in CA1, caused a delayed-onset potentiation
for the recorded field-EPSP and POP in the CA1 region in response to test stimulation of the contralateral CA3. This delayedonset potentiation was dependent on the synergistic activation of
both the glutamatergic and the dopaminergic receptor activation,
because paused glutamatergic test stimulation abolished this potentiation.
TV.07
PROTEOMIC ANALYSIS OF SYNAPTIC PROTEINS
FROM MICE EXPRESSING LOW LEVELS OF p25
Engmann O.1, Thompson A.1, Ward M.2, Giese K.1
1
Institute of Psychiatry, 2 Proteome Science Plc., Kings College
London, London, UK
Cdk5 is a neuronal kinase involved in synaptic plasticity and
memory formation. When overactive, Cdk5 can induce cell
cycle arrest, tau hyperphosphorylation and apoptosis. This
dichotomy in function is correlated with the degree of Cdk5
activation by small regulators. P25 is the most potent activator
of Cdk5, absent in neurons under physiological conditions but
induced during neuronal insults. In mouse models, high levels of p25 lead to neurodegeneration. Consistently, aged mice
with life long exposure to low p25 levels exhibit tau hyperphosphorylation. However, expression of low p25 levels can
improve learning in a sex-specific manner in young adult mice.
The underlying molecular mechanisms of this dose effect are
still poorly understood. Therefore, in the project presented we
have undertaken comparative proteomics on hippocampal synaptosomes from wildtype and p25 mutant mice of both sexes.
Four different approaches of tandem mass tag labeling were
employed. From more than 500 quantifiable proteins, we identified sex-specific changes in the p25 transgenics compared to
wildtype mice. Selected proteins will be introduced and implications for the role of Cdk5-p25 in memory and neurodegeneration will be discussed. This work was supported by an MRC
PhD studentship.
TV.08
INDUCTION OF LONG-TERM POTENTIATION
AND DEPOTENTIATION BY THETA-FREQUENCY
STIMULATION IN THE CA1 FIELD OF RAT
HIPPOCAMPUS IN VITRO
Denisov A.
Biophysics Department, Belarus State University, Minsk, Belarus
One of the most important features of hippocampal long-term potentiation (LTP) is that it is elicited by naturally occurring patterns
of neuronal activity. Long term depression of synaptic transmission in hippocampus is induced by low-frequency stimulation
protocols that completely different from those required for LTP
induction. Depotentiation is another form of synaptic plasticity
that has been hypothesized as a candidate for LTP counterpart in
learning processes. Depotentiation can be induced by theta-frequency stimulation patterns similar to those required for the LTP
induction. We showed that short stimulating sequences applied at
theta frequency to Schaffer collaterals can produce complex population spikes in the CA1 field of rat hippocampal slices if they were
preincubated in modified artificial cerebrospinal fluid. Such thetastimulation induces LTP if the complex spikes emerge. In these
conditions, associative LTP is also produced if weak and strong
pathways are stimulated synchronously. We found that associative LTP induced by synchronous theta stimulation is reversed after application of asynchronous theta stimulation. Thus, opposite
changes of the efficacy of synaptic transmission can be induced by
similar stimulation protocols. Our results confirm the hypothesis
that in some conditions depotentiation may underlie learning rules
in the CA1 field of hippocampus.
TV.09
MATRIX METALLOPROTEINASE-9 AT THE
NEUROMUSCULAR JUNCTION
Yeghiazaryan M.1, Leszczynska A.1, Lasiecka Z.1, Slawinska U.1,
Kaczmarek L.2, Wilczynski G.1
1
Matrix metalloproteinases (MMPs) are considered to play a pivotal role in remodeling of the extracellular matrix in health and
disease. Recent studies suggest that MMP-9 plays an important
role in synaptic plasticity, learning and memory. It was shown
that MMP-9 occurs in postsynaptic part of hippocampal synapses, where the amount and activity of MMP-9 were increased
by stimulation leading plasticity changes. At the neuromuscular
junction, a role, related to the turnover of agrin, has been demonstrated for MMP-3. Although MMP-9 has been localized to NMJ,
neither its role nor precise distribution in relation to synaptic elements is established unequivocally. Here, we have carried out the
investigation of the gelatinolytic activity of NMJ in rats, in order
to establish whether high-intensity exercise can promote expression of MMP-9 at the NMJ. Our fi nding of the present work is
that training increases the gelatinolytic activity of the NMJ in
the extensor digitorium longus and soleus muscles. The 4-week
endurance training program elicited alterations at the presynaptic
side of the NMJ, where the increasing gelatinolytic activity was
localized in the Schwann cells. These data shows that endurance
training influences on the gelatinolytic activity of NMJ, possibly
through synaptic transmission.
334
TV.10
CD44-ErbB RECEPTOR INTERACTION STUDIED BY
FLUORESCENCE RESONANCE ENERGY TRANSFER
ANALYSIS USING FLUORESCENCE LIFETIME
IMAGING MICROSCOPY
Wlodarczyk J.1, Gorlewicz A.2, Wilczek E.3, Gawlak M.2,
Cabaj A.4, Majczynski H.2, Nestorowicz K.5, Herbik M.5,
Grieb P.5, Slawinska U.2, Kaczmarek L.1, Wilczynski G.2
1
of Neurophysiology, Nencki Institute of Experimental Biology
PAS, Warszawa, Poland; 3 Department of Pathology, Medical
University of Warsaw, Warszawa, Poland; 4 Department of
Bionics, Institute of Biocybernetics and Biomedical Engineering,
Warszawa, Poland; 5 Department of Experimental Pharmacology,
CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues.
CD44 was implicated in the development of peripheral nerves,
functioning as a coreceptor for ErbB class of growth factor receptors. However, it is not known whether CD44-ErbB interaction may
occur at the adult peripheral synapses. Here we studied, using Fluorescence Lifetime Imaging Microscopy, the proximity between
CD44 and ErbB3 at the rat neuromuscular junction (NMJ). This
was performed in muscle sections co-immunostained for CD44
and ErbB3, using secondary antibodies coupled to Alexa488 and
Alexa647 respectively. Neuromuscular junctions were visualized
using Alexa555-bound α-BT. The FRET between Alexa488 (donor) and Alexa647 (acceptor) was judged by measuring an accompanying changes in the donor fluorescence lifetime. We found that
the mean fluorescence lifetime of the donor fluorophore labeling
CD44 protein was considerably shorter over the NMJ than in nonsynaptic sites. Then we compared normal rat muscle to the muscle
affected by denervation in the transgenic model of amyotrophic
lateral sclerosis (ALS). Importantly, ALS-like neurodegeneration
resulted in significant increase in molecular proximity of CD44
and ErbB3 at the NMJ. The specific complex formation between
the two proteins was confirmed using immunoprecipitation analysis. Our study provides novel data on the molecular architecture of
the neuromuscular synapse in both health and disease.
TV.11
DYNAMICS OF CORTICAL REMODELING DURING
REMOTE MEMORY STORAGE
Aceti M.1, Vetere G.1, Ammassari-Teule M.2, Restivo L.3
1
Department of Psychobiology, S. Lucia Foundation IRCCS, Rome,
Italy; 2 Department of Psychobiology, CNR Institute for Neuroscience,
Padova, Italy; 3 Department of Neurosciences and Mental Health,
Hospital for Sick Children Research Institute, Italy
There is evidence that reorganization of neuronal circuits in the
anterior cingulate cortex (aCC) occurs during remote memory
storage. Here we describe how morphological changes progressively develop on aCC neurons during formation and stabilization of an aversive memory trace. Using a contextual fear conditioning protocol, we measured memory performance and spine
density on aCC pyramidal neurons at several time points (1, 5,
7, 14, 21 days) following the training episode. Results show that
aversive memory, estimated by the percentage of time spent
freezing, was robust already 1 day posttraining and remained
noteworthy stable until day 21. Morphological measurements in
the trained animlas then revealed no evidence of cortical remodeling on day 5 posttraining, but a significant increase in spines
on day 7 which persisted until day 21. Interestingly, despite an
equivalent number of spines was counted in aCC on days 7, 14,
and 21, the number of single neurons showing increased spine
density strongly varied across time-points. These fi ndings suggest that long term storage of the memory trace requires a relatively stable increase in spines provided by a variable number of
recruited neurons.
TV.12
EVIDENCE FOR CROSS-MODAL PLASTICITY
IN ADULT MOUSE VISUAL SYSTEM
Van Brussel L., Gerits A., Arckens L.
Department of Biology, K.U. Leuven, Leuven, The Netherlands
Since the pioneering work of Hubel and Wiesel in the ’60, research in cat, monkey and humans has led to a central dogma that
beyond the critical period the brain only retains a reduced capacity for reorganization. Current work in rodents however challenges this view since specific treatments are capable of reinstating cortical plasticity in the adult. The goal of our work was to
study this adult cortical reorganization in detail. A combination
of deprivation of one eye and stimulation of the remaining eye
previously led to the identification of input-specific subdivisions
(Van Brussel et al. 2009). Using this information as a reference
map, we established to what extent each of these functional subdivisions take part in cortical reorganization upon enucleation.
Briefly, there seemed to be two waves of recovery, the fi rst characterized by the expansion of the supragranular binocular zone
of V1 and V2L and the second affecting the infragranular layers,
initiated at the outer border of the visual cortex with neighboring
non-visual cortex and accompanied by hyper-activity of this adjacent cortex. To test a possible non-visual nature of this recovery, we combined monocular enucleation with the inactivation
of the remaining eye or a second sensory modality. Both lack
of complete visual cortex deactivation upon enucleation of the
remaining eye, and strong effects of auditory and somatosensory
deprivation on infragranular visual cortex suggest cross-modal
plasticity in adult mice.
TV.13
NEUROPLASTICITY IN THE HIPPOCAMPUS AND THE
STRIATUM OF THE MOUSE DURING VISUO-MOTOR
LEARNING: ANALYSIS OF IMMEDIATE EARLY GENE
EXPRESSION PATTERNS
Laeremans A.1, Nys J.1, Gantois I.2, Arckens L.1, D’Hooge R.2
1
Department of Biology, 2 Department of Psychology, K.U. Leuven,
Leuven, The Netherlands
Visuo-motor learning, a dynamic process in which visual stimuli are associated with certain motor responses, leads to goaldirected, complex behaviours. Literature states that the striatum
and the hippocampus are two important brain areas for this type
of learning since neural mechanisms and changing activation
patterns within these areas are involved in the processing of visual stimuli and the subsequent motor act. The neural activity
of mice trained in the Morris water maze was molecularly char-
acterized by in situ hybridization for the immediate early gene
activity markers arc and homer1a and this in two timeframes,
namely the early learning and the late learning (overtrained/automatisation) phase. First results show that in the early learning
phase arc mRNA expression appears to be upregulated in the
ventrolateral striatum, while in the overtrained phase a higher
expression is noticeable in the dorsal striatum. Arc and homer1a
mRNA expression levels in the hippocampus indicate that this
structure is more active in the early learning phase compared
to the overtrained phase. Subregional analysis of hippocampal
homer1a mRNA expression patterns implies an opposite dynamic of CA1/DG and CA3 in response to learning. While CA1/DG
activity decreases when mice become overtrained, CA3 activity
seems to slightly increase. Further analysis will reveal how differential molecular activity in the subregions of hippocampus
and striatum relates to both phases of the visuo-motor learning
process.
TV.14
PROTEOMIC DISCOVERY OF MATRIX
METALLOPROTEINASE-9 SUBSTRATES
Bajor M., Michaluk P., Kaczmarek L.
Elucidation of protease substrates (“proteodegradomes”) is essential for understanding the proteolytic pathways and their
networks and thus their role in the regulation of cell function.
Matrix metalloproteinase-9 (MMP-9) is expressed by the adult
brain and released in response to enhanced neuronal activity.
It is well established that MMP-9 is involved in neuronal plasticity, including long-term potentiation, learning and memory.
Under pathological conditions, during excitotoxicity, stroke and
traumatic brain injury, MMP-9 is detrimental to the brain tissue, probably because of its enhanced activity. MMP-9 is locally
inhibited by endogenous tissue inhibitors of metalloproteinase 1
(TIMP-1). In the current studies we optimized the isolation of
synaptoneurosomal fractions from the murine hippocampus. In
order to induce MMP-9 activity the synaptoneurosomal fractions
were treated with 50 μM of glutamate for 20 min. To identify
MMP-9 substrates, we compared the synaptic fractions isolated
from wild type and MMP-9 knockout mice by two-dimensional
electrophoresis (2-DE). We have found the differences in the 2-D
gel patterns. Further studies will be complemented by in-gel digestion of the protein spots of interest, mass spectrometry of the
resultant peptides, and peptide mass fi ngerprinting to identify
each protein.
TV.15
HIGHER- ORDER NUCLEAR STRUCTURE
IN EPILEPTOGENESIS
Szczepankiewicz A.1, Walczak A.1, Szczepinska T.2,
Broszkiewicz M.1, Pawlowski K.2, Wilczynski G.1
1
Although the molecular mechanisms of gene-expression in
neurons are comprehensively described in the literature, little
is known about the relationship between these processes and
the architecture of the neuronal cell nucleus. For example, it
has never been examined whether bursts of transcriptional
activity associated with seizures involve any regulation at the
level of higher-order nuclear structure. Accordingly, we have
performed studies on the structure of neuronal nucleus in epileptic animals. Using electron microscopy, we found the striking appearance of large interchromatin granule clusters (IGCs)
in epileptic nuclei. We confi rmed this observation by immunofluorescence-confocal analysis of IGC marker, a spliceosome
assembly factor SC-35. Interestingly, there was also aggregation of spots immunoreactive for phophorylated and acetylated
Histone H3, a marker of transcriptionally active chromatin.
The fi ndings are consistent with the results of bioinformatic
analysis of transcription profiling in the rat kainate-induced
status epilepticus (public gene expression data), showing tendency for coordinated expression of positional gene-clusters
along chromosomes. Taken together, our results suggest that
upon epileptogenesis there is prominent reorganization of neuronal nucleus, putatively involving formation of molecular factories, where transcription, splicing, and export of pre-mRNA
are orchestrated.
TV.16
LOCOMOTOR EXERCISE OF SPINAL RATS CAUSES
AN INCREASE IN SIZE OF CHOLINERGIC TERMINALS
IN EXTENSOR MOTOR NUCLEI
Czarkowska-Bauch J.1, Mankovskaya T.1, Zaremba M.1,
Gajewska O.1, Ziemlinska E.1, Platek R.1, Mlodkowski M.2,
Korczynski J.3, Skup M.1
1
Biology PAS, Warszawa, Poland; 2 Wikom, Wikom Ltd., Warszawa,
Poland; 3 Laboratory of Confocal Microscopy, Nencki Institute of
Spinal cord transection causes dramatic, sustained decrease of
vesicular acetylcholine transporter VAChT in terminals contacting motoneurons, as reported by Kitzman (2006, Exp Neurol
197). Cholinergic projection is known to regulate excitability
of motoneurons during locomotion. The question arises if locomotor exercise of spinal, paraplegic animals might restore the
role of this projection. Three groups of adult rats were tested:
intact control (n=6), spinal (n=7) and spinal trained subjected
to 5 weeks of treadmill locomotor training (n=8). Animals were
spinalized at low thoracic segments. Gastrocnemius/soleus and
anterior tibial motoneurons were prelabeled with fluorescent
dyes (FG, DY), injected to the muscles. VAChT immunoreactivity (IR) was detected using polyclonal Sigma antibody. We
have found that the spinal cord transection caused a decrease
of VAChT IR in boutons synapsing on cell bodies and proximal dendrites of motoneurons in L3 and L4 segments compared
with that of intact rats. Surprisingly, training caused its further
decay. Mean number of VAChT IR boutons did not differ consistently between groups. However, in the extensor motoneuron
pools of trained animals the number of bigger VAChT IR boutons was clearly higher than in spinal non-trained and intact
animals. The latter effect is in line with functional improvement
of spinal trained animals, which is the most prominent in the
support phase of locomotion. Supported by MSE P-N/029/2006
and N N401 0480 33 grants.
336
TV.17
MONOCULAR ENUCLEATION INDUCES PROMINENT
THYMOSIN BETA 4 EXPRESSION IN MICROGLIA AND
NEURONS OF THE SUPERIOR COLLICULUS OF ADULT
MICE
Paulussen M., Verleden S., Arckens L.
Laboratory of Neuroplasticity and Neuroproteomics, K.U. Leuven,
Leuven, The Netherlands
Thymosin beta 4 (Tβ4) is a peptide of 43 amino acids, mainly recognized as a regulator of actin polymerisation by sequestering Gactin. The peptide has been implicated in apoptosis, angiogenesis
and lesion-induced neuroplasticity. Tβ4 is widely distributed in the
brain, in neurons as well as in microglia. In this study we examined possible changes in Tβ4 expression in the visual system of the
mouse after surgical removal of one eye. Adult mice were deprived
of vision and sacrificed 1, 3 and 5 days and 1, 2 and 7 weeks after
enucleation of the right eye. After 3 days of monocular deprivation,
a substantial up-regulation of Tβ4-positive microglia was visible in
the left superior colliculus, which faded with post-lesion survival
time. The Tβ4-positive microglia appeared activated for clearance
of debris of degenerating axons and myelin. Apart from this microglial activation, a remarkable neuronal response was observed.
In the deeper layers of the left and right superior colliculus Tβ4positive neurons with very long Tβ4-positive neurites became apparent. These cells were visible within 1 week but became more obvious 7 weeks post-lesion. Tβ4 may therefore participate in growth
or remodeling of neuronal processes in deep layers of the superior
colliculus possibly to induce some form of recovery of the visual
system upon sensory deprivation.
TV.18
THE INVOLVEMENT OF GLUCOCORTICOID AND
MINERALOCORTICOID RECEPTORS IN THE EFFECTS
OF BRIEF RESTRAINT STRESS ON LTP AND LTD IN
THE DENTATE GYRUS OF MICE
Spyrka J.1, Danielewicz J.2, Hess G.1
1
Institute of Pharmacology PAS and Institute of Zoology Jagiellonian
University, Kraków, Poland; 2 Institute of Zoology Jagiellonian
Long-term potentiation (LTP) and long-term depression (LTD)
are two cellular models of synaptic plasticity. These phenomena
can be modulated by stress. Stress is correlated with an increased
release of glucocorticoids (GCs) from the adrenal cortex. GCs act
via two receptor types, glucocorticoid receptors (GRs) and mineralocorticosteroid receptors (MRs) which are expressed in a high
density in the dentate gyrus (DG). Here we studied whether single
brief restraint stress affects LTP and LTD in the DG and if these
effects are mediated by GR and MR receptors. C57BL/6 male
mice were subjected to the restraint, lasting 10 minutes. 1 hour
before immobilisation animals were subcutaneously injected with
glucocorticoid receptor antagonist RU38486 or mineralocorticoid
receptor antagonist spironolactone. Hippocampal slices were prepared immediately after the end of restraint. Field excitatory postsynaptic potentials (fEPSP) were evoked in the molecular layer of
the DG by the stimulation of the lateral perforant path. LTP was
induced by 4 trains of high-frequency stimulation (100 Hz, 1 s, repeated every 25 s). LTD was evoked by low-frequency stimulation
(1 Hz, 15 min). In slices from stressed mice LTP was enhanced,
but LTD was impaired. The data indicate that facilitatory effects
of restraint on LTP are diminished by RU38486 and spironolactone indicating the involment of both GRs and MRs. Preliminary
results suggest that the inhibitory effects of restraint on LTD are
decreased by RU38486.
TV.19
THE INVOLVEMENT OF MINERALOCORTICOID
RECEPTORS IN THE EFFECTS OF REPEATED BRIEF
RESTRAINT STRESS ON LTP IN THE DENTATE GYRUS
OF MICE
Spyrka J., Hess G.
Institute of Pharmacology PAS and Institute of Zoology Jagiellonian
Increased level of corticoid hormons during stress results in activation of hippocampal mineralocorticosteroid receptors (MRs)
and glucocorticoid receptors (GRs). Our earlier studies showed
impairment of LTP in the dentate gyrus (DG) of mice after 3 daily sessions of brief (10 min) restraint stress and LTP augmentation after 14 and 21 daily sessions of immobilization. In contrast,
1 and 7 daily sessions of immobilization did not affect LTP level.
We demonstrated that the GR antagonist RU38486 selectively
blocks the effect of 3 sessions of restraint. In the present study we
investigated whether the effects of repeated brief restraint stress
are mediated by MRs. C57BL/6 male mice were exposed to the
restraint, lasting 10 minutes, for 3 and 14 days. 1 hour before immobilization the animals were subcutaneously injected with the
MR receptor antagonist spironolactone. Mice were sacrificed 24 h
after the last exposure to restraint and hippocampal slices were
prepared. Field excitatory postsynaptic potentials were evoked
by the stimulation of the lateral perforant path and recorded from
the molecular layer of the DG. LTP induction was attempted by
applying 4 trains of high-frequency stimulation (HFS, 100 Hz,
1 s, repeated every 25 s). In both experimental groups immediately after HFS recorded responses were potentiated but 2 h later
they were diminished relative to control. Thus, the present results
show that spironolactone blocks LTP induction after 3 and 14
days of daily restraint.
TV.20
IDENTIFICATION OF CELLS UNDERGOING PLASTIC
CHANGES IN THE BARREL CORTEX AFTER SIMPLE
ASSOCIATIVE LEARNING USING TRANSGENIC
fosGFP MICE
Garkun Y.1, Bekisz M.2, Radwanska A.1, Barth A.3, Kossut M.1
1
Warszawa, Poland; 3 Department of Biological Sciences, Carnegie
Mellon University, Pittsburgh, USA
Learning is assumed to be connected with neuronal c-fos expression. In this study we investigate whether associative learning involving tactile stimulation of one row of whiskers induced changes
in c-fos expression level in neurons within the cortical representation of the stimulated vibrissae. We use transgenic mice in which
the expression of green fluorescent protein (GFP) is controlled by
c-fos promoter [Barth et al. (2004) J Neurosci]. This construct allows us to identify individual neurons undergoing plastic changes
in acute (living) brain slices using standard fluorescence imaging.
In the trained mice prior to experiments a 3-day sensory stimulation (20 min/day) of row B of whiskers (conditioned stimulus)
paired with an electrical shock (unconditioned stimulus) was performed. The second group comprised of untrained animals. Acute
slices from the somatosensory cortex (cut orthogonally to the rows
of barrels) were prepared after the end of training and investigated
using upright fluorescent microscope. Our preliminary data indicate high variability of fosGFP expression throughout cortical layers. Biggest amount of GFP-labeled cells are observed in layer 2/3
and much smaller in layers VI and V. Sensory training alter amount
of fosGFP+ cells in layer 2/3 of barrel B as compared to other barrels with little or no effect in layers IV and V. Supported by the
Ministry of Science and Education grant: N30308131/2682.
TV.21
ALTERATIONS IN SYNAPTIC PLASTICITY CAUSED
BY ST. JOHN’S WORT MAY MITIGATE NEGATIVE
EFFECTS OF STRESS ON SPATIAL WORKING
MEMORY
Trofimiuk E., Braszko J.
Department of Clinical Pharmacology, Medical University of
Białystok, Bialystok, Poland
St. John’s wort (Hypericum perforatum) is one of the popular herbal
drugs in Europe and USA. We have recently described beneficial
effects of this herb in the treatment of stress-evoked memory impairments. The aim of the present study was to test a hypothesis that
St. John’s wort alleviates stress- and corticosterone-related memory
impairments by restoring levels of synaptic plasticity proteins: neuromoduline (GAP-43) and synaptophysin (SYP) in hippocampus
and prefrontal cortex. Stressed and corticosterone treated rats presented a significantly delayed acquisition of spatial working memory (P<0.001) in the Barnes maze (BM). Chronic administration of
H. perforatum (350 mg kg-1 for 21 days) potently and significantly
improved processing of spatial information in the stressed and corticosterone treated rats (P<0.001). Also the herb increased levels of
GAP-43 and SYP, in both St. John’s wort treated rat’s hippocampus
(P<0.05 and P>0.05, respectively) and prefrontal cortex (P<0.05
and P<0.05) as measured by western immunoblotting. We found
that H. perforatum prevented the deleterious effects of both chronic
restraint stress and prolonged corticosterone treatment on working
memory measured in BM test. These findings indicate that at least
part of the beneficial effect of H. perforatum on memory may be
mediated by GAP-43 and SYP proteins.
TV.22
MMP-9 ACTIVATION AFTER NEURONAL
STIMULATION IS A POLYADENYLATION DEPENDENT
PROCESS
Dziembowska M., Janusz A., Romanowska E., Kaczmarek L.
Laboratory of Molecular Neurobiology, Nencki Institute of
Recent studies indicate that MMP-9 (gelatinase B that regulates
pericellular environment through the cleavage of protein components of the extracellular matrix) plays a role in synaptic plasticity.
Szklarczyk et al. (2002), Konopacki et al. (2008) and Wilczynski et
al. (2008) have demonstrated the presence of mRNA and protein for
MMP-9 at postsynaptic sides of rat hippocampal neurons. It was also
shown by Michaluk et al. (2007) that gelatinolytic activity of MMP9
increases after stimulation of rat neuronal cultures with either glutamate or bicuculine. We observed the presence of MMP-9 protein and
mRNA in synaptoneurosomes, the synaptic fraction isolated from
hippocampus. To detect the activity of MMP-9 we measured the
cleavage of its substrate, β-dystroglycan. By the use of this readout
we showed that MMP-9 is activated 5 to 10 mintes after neuronal
stimulation. We postulate here that MMP-9 is translated from dendritically-localized mRNA and the protein is produced in response to
synaptic stimulation. Its rapid and local translation and secretion is a
polyadenylation- and local translation-dependent process.
TV.23
EPIGENETIC REGULATION OF MATRIX
METALLOPROTEINASE-9 TRANSCRIPTION DURING
EPILEPTOGENESIS
Zybura K., Amborska R., Kaczmarek L., Rylski M.
Matrix Metalloproteinase 9 (MMP-9) plays an important role in a
physiological and aberrant synaptic plasticity, like that occurring
during epileptogenesis. Consequently, enhanced synaptic plasticity
is accompanied by a prolonged upregulation of MMP-9 expression
due to an activation of its transcription. Epigenetic mechanisms are
often responsible for a long lasting changes in a gene expression.
Accordingly, our recent data suggested that MMP-9 gene transcription in brain neurons can be regulated by these molecular phenomena. Here, we have studied epigenetic changes occurring in a Mmp9 promoter in the rat hippocampus during PTZ-induced kindling,
which is a model of an epileptogenesis. Using the methylationspecific polymerase chain reaction, we have evaluated pattern of
alterations of the methylation status in the 5’CpG sites of the Mmp-9
promoter which arise during different stages of the kindling. Then,
we have confirmed these data with the bisulfite sequencing.
TV.24
SPATIO-TEMPORAL CHARACTERIZATION OF mTOR
KINASE ACTIVITY INDUCED BY KAINIC ACID
EVOKED SEIZURES
Macias M., Blazejczyk M., Jaworski J.
The common effects of epileptic seizures are aberrant plastic
changes in synaptic connectivity and morphological rearrangements in hippocampal mossy fibers. This phenomenon is believed
to be a basis of facilitation of further epileptic episodes, however its
molecular mechanism remains elusive. mTOR kinase is crucial for
rearrangements of neuronal morphology and for long term synaptic
plasticity. Overactivation of mTOR was reported in brain pathologies associated with seizures (tuberous sclerosis, cortical dysplasia). Experimental evidence supports causative role of mTOR in
these forms of epilepsy. So far, however, links of mTOR signaling
pathway to other types of epilepsy are unknown. To address this issue we investigated activation of mTOR and its downstream effectors in rat brain after kainic acid (KA) induced epilepsy. Animals
were sacrificed 2, 6, 12, 24 and 72 hours after KA administration
and levels of phosphorylated mTOR and S6 has been examined.
Both, biochemical methods and immunohistochemistry proved S6
activation in hippocampus and cerebral cortex at all time points after KA. Immunohistochemical analysis additionally revealed that
338
2 h after KA, activities of mTOR and S6 are upregulated mainly in
neurons while after 6 h there was also enhancement of glial S6 immunoreactivity. Our data suggest that KA induced seizures activate
mTOR signaling in various cell types with specific spatio-temporal
pattern. This work has been financed by PNRF-96-AI-1/07 grant.
TV.25
STIMULATION OF THE VENTRAL TEGMENTAL AREA
INDUCES HIPPOCAMPAL THETA RHYTHM AND
c-fos EXPRESSION IN THE RAT BRAIN
Majkutewicz I., Kusmierczak M., Blajet M.,
Orzel-Gryglewska J., Jurkowlaniec-Kopec E.
Poland
Our previous study indicated that microinjection of procaine or
electrolytic lesion of the ventral tegmental area (VTA) suppressed
hippocampal theta rhythm in urethane-anaesthetized rats. The aim
of this study was to verify the hypothesis that electrical stimulation
of the VTA induces hippocampal theta rhythm and to find brain
structures particularly active during this phenomenon and probably
involved in its mechanism. The study was performed on urethane
anaestethized male Wistar rats with an electrode implanted unilaterally in the VTA or zona incerta (ZI – control group). Stimulation
was applied as 0.1-ms rectangular impulses of 50 Hz frequency and
duration of 30 s at 10-min intervals. VTA stimulation within the
current intensity range of 100–240 mA evoked hippocampal theta
rhythm, manifested as synchronization of the EEG signal and an
increase in the power at 3–6 Hz band. ZI stimulation did not elicit
such effects. After VTA stimulation we also found induction of
c-fos expression in brain regions connected to the VTA: nucleus
accumbens, lateral septum, or engaged in the regulation of hippocampal theta rhythm: medial septum, midline thalamic nuclei,
hypothalamic nuclei, pedunculopontine, laterodorsal and cuneiform
tegmental nuclei. The results indicate that the VTA may be a part
of the brainstem theta synchronizing system and may influence the
hippocampal EEG through indirect pathway via hypothalamus and
the medial septum, simultaneously increasing thalamic activity.
TV.26
EFFECT OF EXTRA HIGH POWER PULSES AND
HYDROGEN PEROXIDE ON FUNCTIONAL ACTIVITY
OF NEURONS
Hakobyan K.1, Hunanyan A.2, Ghazaryan A.1, Ayrapetian S.1
1
Department of Neuroscience and Pain, UNESCO Chair-Life
Sciences International Postgraduate Educational Center, Yerevan,
Armenia; 2 Department of Neurobiology and Behavior, SUNY
at Stony Brook, Stony Brook, NY, USA
It was recently shown that the exposure of aqueous solutions to highpeak-power microwave pulses leads to the formation of hydrogen
peroxide (H202). The aim of this study was to evaluate the effects of
extra high power pulses (EHPP,9.3 GHz, 101.2 kW/kg) millimeter
waves and H202 on action potentials, acetylcholine chemosensitivity and cell volume of giant neurons from the parietal ganglion of
Helix Pomatia by means of standard voltage-clamp method. H202
containing and EHPP treated physiological solutions increased the
interburst period of neuronal pacemaker activity. In K-free physiological solution (when Na+/K+ pump is inactive) the interburst period
was higher than in normal one. Application of H202 containing and
EHPP treated physiological solutions changed the number of active
receptors on neuromembrane surface which caused depression of
its chemosensitivity and decreased as a consequence of cell volume
(cell shrinking). These comparative studies allowed us to suggest
that EHPP induced formation of H2O2 is one of the main messenger
through which the non-thermal biological effect of EHPP on functional activity of neurons is realized.
TV.27
SINGLE AND ENSEMBLE NEURAL VARIABILITY
WITH SERIAL CORRELATION
Farkhooi F., Nawrot M.
Department of Neuroinformatics and Theoretical Neuroscience,
FU Berlin, Berlin, Germany
The activity of spiking neurons is frequently described by renewal
point process models. However, the assumption of independent intervals must be questioned for many different types of neurons. We
review experimental studies that reported the feature of a negative
serial correlation of neighboring intervals, commonly observed in
neurons in the sensory periphery as well as in central neurons, notably
in the mammalian cortex. In our experiments we observed the same
short-lived negative serial dependence of intervals in the spontaneous activity of mushroom body extrinsic neurons in the honeybee.
To model serial interval correlations of arbitrary lags, we suggest a
family of autoregressive point processes. Its marginal interval distribution is described by the generalized gamma model, which includes
as special cases the log-normal and gamma distributions, which have
been widely used to characterize regular spiking neurons. In numeric simulations we investigated how serial correlation affects the
variance of the neural spike count. We show that the experimentally
confirmed negative correlation reduces single-neuron variability, as
quantified by the Fano factor, by up to 50%, which favors the transmission of a rate code. We argue that the feature of a negative serial
correlation is likely to be common to the class of spike-frequencyadapting neurons and that it might have been largely overlooked in
extracellular single-unit recordings due to spike sorting errors.
TV.28
MODIFICATION OF EXCITABILITY OF CORTICAL
NEURONS INDUCED BY ASSOCIATIVE LEARNING
– EX VIVO EXPERIMENTS
Bekisz M.1, Garkun Y.2, Hess G.3, Wrobel A.1, Kossut M.2
1
Cellular Neurobiology, Nencki Instituite of Experimental Biology
PAS, Warszawa, Poland; 3 Department of Physiology, Institute of
Pharmacology, PAS, Kraków, Poland
In adult mice, whiskers stimulation paired with an electrical shock
to the tail induces expansion of the cortical representation of stimulated vibrissae and enhances inhibitory interactions within the
“trained” barrels. In present experiments we investigated whether
such simple associative learning paradigm induced also changes in
the neuronal excitability. We prepared brain slices through the barrel cortex and performed whole-cell recordings from visually identified layer IV neurons. Our results showed that excitatory cells located in layer IV of the cortical representation of the “trained” row
B of vibrissae had higher frequency of spikes recorded at threshold
potential, as compared to both neurons from “untrained” barrel D
and to cells from control animals. Additionally, cells within the
“trained” barrels displayed bigger gain in the input-output function and decrease in the activation of BK channels responsible for
the fast afterhyperpolarisation, which suggests the source for their
enhanced intrinsic excitability. The increased excitability of cells
within the “trained” barrels may express their homeostatic plasticity which parallels enhanced inhibitory interactions found previously within layer IV of the cortical representation of the “trained”
vibrissae. This may be the way to increase the cortical selectivity of
response to sensory input from “trained” whiskers. Supported by the
MNiSW grants: N30308131/2682, N40114631/3239, COST/127/2007
and PBZ/MNiSW/07/2006.
TV.29
ELUCIDATING THE FUNCTION OF CPEB PROTEINS
IN MICROGLIA
Kaczmarczyk L.1, Turimella S.1, Vangoor V.1, Seifert G.1,
Dublin P.1, Neumann H.2, Steinhäuser C.1, Theis M.1
1
Institute of Cellular Neurosciences, 2 Institute of Reconstructive
Neurobiology, University Bonn LIFE & BRAIN Center, Bonn,
Germany
Cytoplasmic Polyadenylation Element Binding proteins (CPEBs)
modulate local protein synthesis in neurons crucial for LTP maintenance, memory, and learning. Our recent results indicate they are
involved in the progression of epilepsy in a mouse model of temporal
lobe epilepsy (TLE). Neuronal proteins regulated by CPEBs include
tissue plasminogen activator (tPA), but the primary source of tPA in
brain is microglia. CPEB function in glial cells has hardly been investigated. Since microglial tPA is a crucial neurodegenerative factor in TLE, we explore the attractive hypothesis of microglial CPEB
involvement in tPA release and, thereby, neurodegeneration following
seizures. By single cell RT-PCR we observed CPEB1-4 mRNAs in
mouse microglia. This corroborates RT-PCR and immunoblot results,
showing abundant CPEB transcript and protein levels in the BV-2 microglial cell line and ES-cells-derived microglia. We use CX3CR1
EGFP/+ knock-in transgenic mice to isolate microglial cells by FACS.
In such primary microglia cultures, we will compare CPEB and tPA
expression, polyadenylation state of tPA mRNA, and phosphorylation status of CPEBs before and after microglial activation. Finally,
we will look at CPEB-mediated control of tPA expression with a luciferase reporter assay. We hope the abovementioned studies to shed a
new light on the function of microglial CPEBs and their potential role
in neurodegeneration associated with TLE.
TV.30
FACILITATED EXTRACELLULAR DIFFUSION IN THE
CORPUS CALLOSUM OF BRAL1-DEFICIENT MICE
Dmytrenko L.1, Vargova L.1, Vorisek I.2, Bekku Y.3, Oohashi T.3,
Sykova E.2
1
Department of Neuroscience, Charles University 2nd Medical
Faculty, Prague, Czech Republic; 2 Department of Neuroscience,
Institute of Experimental Medicine AV CR, Prague, Czech
Republic; 3 Department of Molecular Biology and Biochemistry,
Okayama University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Okayama, Japan
The extracellular matrix molecule complex stabilized by Bral1 at
the nodes of Ranvier might play a role in neuronal conduction as this
complex represents a reservoir and/or diffusion barrier for Na+ ions.
We measured the apparent diffusion coefficient of water (ADCW) by
diffusion-weighted MRI and the extracellular space (ECS) diffusion
parameters volume fraction α (α = ECS volume/total tissue volume)
and tortuosity λ (λ2 = free/apparent diffusion coefficient) by the real-
time iontophoretic method. Measurements were done in the somatosensory cortex and along the mediolateral (x), rostrocaudal (y) and
ventrodorsal (z) axes in the corpus callosum (CC) of Bral1 +/+ and
-/- mice. In the cortex, we did not find any significant differences in
the ECS diffusion parameters or ADCW values between Bral1 +/+
and -/- mice. In the CC, λ in Bral1 +/+ mice was significantly higher
along all three axes (λx = 1.41 ± 0.03; λy = 1.70 ± 0.02; λz = 1.72 ±
0.01; mean ± SEM) compared to -/- mice (λx = 1.32 ± 0.02; λy = 1.56
± 0.01; λz = 1.58 ± 0.02); there were no significant differences in α.
These data were confirmed by a lower ADCW in the CC of Bral1 +/+
than of -/- mice, along the x (1 158 ± 55 μm2s-1 and 1 340 ± 25 μm2s-1,
respectively) and y axes (442 ± 19 μm2s-1 and 521 ± 24 μm2s-1, respectively). We conclude that the deletion of Bral1 results in a reduction
of diffusion barriers at the nodes of Ranvier, which in turn facilitates
diffusion in all directions in the myelinated white matter.
TV.31
LACK OF NEUROPROTECTIVE EFFECT OF
ASTROGLIA-DERIVED BDNF ON HIPPOCAMPAL CELL
CULTURES EXPOSED TO TRIMETHYLTIN
Figiel I., Wlodarczyk J.
It is well known that in response to neuronal injury activated glial
cells produce pro-inflammatory cytokines and neurotrophic factors. These immunoregulatory molecules may play either neurotoxic or neuroprotective roles. In previous studies on mixed
neuronal-glial cultures of rat hippocampal dentate gyrus we demonstrated that trimethyltin (TMT) induced neuronal cell death accompanied by an enhanced production of tumor necrosis factor
alpha (TNF-α) in microglial cells and a strong increase in TNF-α
receptor 1 (TNFR1) expression on astrocytes. Since evidence has
been provided that TNFR1 could mediate the production of neurotrophic factors, in the current studies we examined whether the
brain-derived neurotrophic factor (BDNF) is up-regulated after
TMT exposure and whether it may exert neuroprotective effect on
dentate granule cells. Using western blot analysis and immunocytochemical staining we have shown a dose-dependent increase in
BDNF production, mainly in astrocytes. Quantitative fluorescence
analysis revealed that addition of anti-TNFR1 antibody to TMTtreated cultures suppresses the astroglial synthesis of BDNF.
Nevertheless, immunocytochemical studies of active caspase-3
demonstrated the high level of its expression in cultures exposed
to TMT, as well as in cultures pre-treated with BDNF. Our data
suggest the involvement of TNFR1 pathway in BDNF production
in astrocytes and indicate that this neurotrophic factor does not
protect granule neurons against TMT injury.
TV.32
INFLUENCE OF ANTIDEPRESSANTS ON CYTOKINES
(TNF-α, IL-1β, IL-10) IN LIPOPOLYSACCHARIDESTIMULATED PRIMARY RAT MIXED GLIAL CELL
CULTURES
Bielecka A.1, Pudelko A.1, Paul-Samojedny M.2, Kowalski J.2
1
Deapartment of Pharmacology, Medical University of Silesia,
Katowice, Poland; 2 Department of Medical Genetics, Medical
University of Silesia, Sosnowiec, Poland
It is suggested that glial activation play an important role in the
pathogenesis of psychiatric and neurodegenerative diseases. Ac-
340
tivated glial cells secrete various cytokines. Anti-inflammatory
effect of imipramine, moclobemide, fluoxetine was investigated
using 13–14 day primary rat mixed glial cultures prepared from
cerebral hemispheres of one-day old newborn Wistar rats. LPSstimulated levels of TNF-α, IL-1β, IL-10 were measured with
ELISA kits in culture medium. Antidepressants were used at concentrations from 108 to 100 μM. mRNA for cytokines was evaluated by RT-QPCR. Moclobemide, fluoxetine (108 to 10 μM) and
imipramine (106 to 100 μM) reduced TNF-α release. IL-1β concentration was diminished by moclobemide, imipramine (106 to
100 μM) and fluoxetine (10-6 to 10 μM) but level of IL-10 was
not changed significantly after drug administration. The levels of
TNF-α or IL-1β mRNA were reduced by the studied drugs (10
μM), whereas IL-10 mRNA level was only attenuated. Our results
support observation that antidepressants have anti-inflammatory
effects in CNS because they affected the balance between proand antiinflammatory cytokines (TNF-α, IL-1β/IL-10) in mixed
glial culture. This work was supported by Grant N 401 130 31/2871
from the Ministry of Science and Higher Education.
TV.33
CELL-TYPE SPECIFIC REGULATION OF MORPHINEAND GLUCOCORTICOID INDUCED GENES
Slezak M., Bilecki W., Gieryk A., Solecki W., Kubik J.,
Przewlocki R.
Single morphine administration to mice leads to changes in the
expression of numerous genes in mouse striatum. Among them
are genes regulated by the glucocorticoid receptor activation,
like Sgk1, Gilz and Plzf [Korostynski et al. (2007) Genome Biol].
Analysis of their expression patterns with the use of in situ hybridization revealed their different regional distribution throughout the
brain. To verify the cellular specificity of expression, the double
immunohistochemical stainings were performed with antibodies
directed against investigated proteins and cellular markers (NeuN,
GFAP, CC1, OX42). The results show that certain morphine- and
GR-induced proteins are expressed in neurons, whereas others in
glial cells. Hence, their regulation depends on the cell-type specific context.
TVI: Disorders of the Nervous System II
TVI.01
DISASSEMBLY OF SHANK AND HOMER SYNAPTIC
CLUSTERS IS DRIVEN BY SOLUBLE β-AMYLOID
THROUGH DIVERGENT NMDAR-DEPENDENT
SIGNALLING PATHWAYS
Roselli F.1, Wegerich Y.1, Defazio G.2, Livrea P.2, Almeida O.3
1
Department of Neuroendocrinology, Max-Planck Institute of
Psychiatry, Munich, Germany; 2 Department of Neurological and
Psychiatric Sciences, University of Bari, Italy; 3 Department of
Neuroendocrinology, Max-Planck Institute for Psychiatry, Munich,
Germany
Disruption of the postsynaptic density (PSD) is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer’s disease. We show that Aβ disrupts two other scaffold proteins,
Homer1b and Shank1. Treatment of cortical neurons with soluble
Ab results in rapid thinning of the PSD, decreased synaptic levels
of Homer1b and Shank1, and reduced synaptic mGluR1 levels; the
latter changes require NMDAR and voltage-dependent calcium
channel (VDCC) activity. We also show that de novo protein synthesis is required for the declustering effects of Aβ on Homer1b (but
not Shank1) and that, in contrast to PSD-95, Aβ-induced Homer1b
and Shank1cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Aβ diverges in
two other respects: (1) whereas the activity of both NMDAR and
VDCC is required for Aβ-induced declustering of Homer1b, Aβinduced declustering of Shank1 only requires NMDAR activity;
(2) whereas the effects of Aβ on Homer1b involve engagement of
the PI-3K pathway and PP2B activity, those on Shank1 involve activation of ERK/RSK. Thus, Aβ recruits discrete signaling pathways
to rapidly reduce the synaptic localization of major components of
the PSD and to regulate the availability of mGluR1.
TVI.02
INVOLVEMENT OF THE CacyBP/SIP PROTEIN
IN TAUOPATHY
Schneider G.1, Mietelska-Porowska A.2, Filipek A.1,
Niewiadomska G.2
1
Warszawa, Poland
The CacyBP/SIP protein is highly expressed in brain neurons,
where it localizes in somata and in neuronal processes. The role
of CacyBP/SIP is not clear at present but recently it has been suggested that the interaction between CacyBP/SIP and tubulin might
be important for stability of microtubules in neuronal cells. Particularly, it was found that CacyBP/SIP changes its localization in
an age-dependent way. Moreover, these changes in localization are
similar to those observed for tau, a well known tubulin binding
protein. Since it is known that changes in localization of tau during aging are similar to those observed in tauopaties (such as Alzheimer’s disease), in this work we examined the localization and
possible role of CacyBP/SIP in mouse model of tauopathy (mice
overexpressing the tau protein). We found that in young tauopathic
mice CacyBP/SIP and tau are mainly present in neuronal somata
whereas in wild type animals both proteins are localized in somata
and in neuronal processes. Moreover, the tubulin staining pattern
in transgenic mice was different than in wild animals. This might
suggest that translocation of tau together with CacyBP/SIP affects
stability of microtubules in neuronal cells. At present we examine
whether posttranslational modification(s) of CacyBP/SIP, such as
phosphorylation, might be responsible for the observed translocation of CacyBP/SIP during tauopathy. This work was supported by
statutory funds from the Nencki Institute of Experimental Biology.
TVI.03
THE TRIPEPTIDE RER AND ITS POTENTIAL ROLE
IN THE AMELIORATION OF SYMPTOMS
IN ALZHEIMER’S DISEASE ANE OTHER DEMENTIAS
Bodo C., Giese K.
Centre for the Cellular Basis of Behaviour, King’s College London,
UK
Alzheimer’s disease (AD) is a neurodegenerative syndrome with
high prevalence in the elderly population and whose early symp-
toms include impairment in spatial memory. Inherited forms of
AD can be caused by dominant, single missence mutations in the
Amyloid Precursor Protein (APP) gene, and this has been used
to generate several animal models for the disease by overexpression of a mutated version of the protein. We have established at
our institute a breeding colony of transgenic mice expressing the
London mutation of APP as a transgene (APP[V717I]), and tested
them at three and six months of age on the Morris Water Maze to
confirm a previous report on their impairment in the acquisition
of this particular task. Moreover, APP[V717I] mice were used as a
model to determine whether treatment with the tripeptide RER, a
compound that has previously been shown to ameliorate amyloidinduced amnesia in young chicks, is capable of modifying their
performance in the maze in a way that compensates the deficits in
spatial memory derived from the expression of the transgene. This
work was supported by Grant #88 provided by the Alzheimer’s
Society.
TVI.04
CELL TYPE SPECIFIC UPTAKE AND CLEARANCE
OF β-AMYLOID PEPTIDES
Kuszczyk M.1,2, Sadowski M.1
1
Department of Neurology, New York University School of Medicine,
New York, NY, USA; 2 Department of Neurochemistry, Mossakowski
Medical Research Centre PAS, Warsaw, Poland
Aggregation of β-amyloid peptides (Aβs) into toxic oligomers,
amyloid fibrils, and its deposition in senile plaques initiate the
neurodegenerative cascade of Alzheimer`s disease. Intraneuronal accumulation of Aβs starts prior to the appearance of senile plaques, and contributes to neurodegeneration by affecting
protein trafficking, mitochondrial metabolism, tau phosphorylation, and synaptic plasticity. The aim of this study was to compare
the uptake and clearance of Aβs by various cell lines. SK-N-SH
neuroblastoma cells rapidly metabolized Aβ clearing 40 μM of
Aβ40 from the medium within 48 h. Pulse-uptake experiment
demonstrated that Aβ40 was totally metabolized inside SK-NSH cells within 6 h. Presence of apolipoprotein E4 accelerated
the Aβ40 uptake. In comparison, primary hippocampal neurons
internalized only small fraction of Aβ40 present in the medium
but showed notable intraneuronal Aβ40 accumulation. In pulseuptake experiment, intraneuronal presence of Aβ40 monomers
and oligomers could be demonstrated after 24 h. Accumulation
of Aβ40 by hippocampal neurons affected cell viability and membrane integrity as determined by MTT and LDH release assays,
respectively. Our results demonstrate that neuroblastoma cells
internalize and metabolize Aβ40 more efficiently than primary
hippocampal neurons. Therefore, under conditions of elevated
Aβ level, hippocampal neurons are susceptible to Aβ accumulation and intraneuronal oligomerization what leads to downstream
toxic effects.
TVI.05
INCREASED VULNERABILITY TO OXIDATIVE STRESS
IN LYMPHOCYTES FROM SAD AND FAD PATIENTS
Bialopiotrowicz E., Kuzniewska B., Kachamakova N.,
Kuznicki J., Wojda U.
Laboratory of Neurodegeneration, International Institute of Molecular
and Cell Biology, Warszawa, Poland
Alzheimer’s disease (AD) is the most common neurodegenerative disorder in which certain molecular changes are observed
not only in neurons but also in peripheral cells. Growing evidence suggests that AD post-mitotic neurons exhibit increased
apoptotic response to oxidative stress, mitochondria dysfunction and calcium dyshomeostasis. We hypothesized that some
of these alterations could be observed in peripheral lymphocytes
and studied for potential diagnostic purposes. We analyzed
apoptotic response to the redox stress evoked by 2-deoxy-Dribose in immortalized lymphocytes from 18 patients with sporadic AD (SAD), from 2 familial AD (FAD) patients with novel
mutations in presenilin 1: P117R and I213F, and from 20 agematched healthy individuals. Using two independent flow cytometry methods for quantification of apoptosis, we found that
SAD and FAD lymphocytes show enhanced apoptotic response
to the redox stress. This apoptotic response was accompanied
by decline in mitochondrial membrane potential measured with
JC-1 as well as by increased activities of caspase 9 and caspase
3. However, no changes in the expression of two calcium-binding proteins: calmyrin 1 and calreticulin were observed. This
study emphasizes that increased susceptibility to redox stress
and associated upregulation of mitochondrial apoptotic pathway
is characteristic not only for AD neurons, but also for AD lymphocytes. Thus, human lymphocytes could be used in further
studies on AD pathogenesis.
TVI.06
RESVERATROL AND NITRIC OXIDE CHOLINERGIC
NEUROTOXICITY
Gul-Hinc S., Bielarczyk H., Jankowska-Kulawy A., Szutowicz A.
Gdańsk, Poland
Several scavengers of free radicals like resveratrol (RV) are
claimed to be useful in complementary therapy of Alzheimer’s dementia (AD) and other cholinergic encephalopathies,
through the protection of mitochondrial energy metabolism.
NO excess is one of recognized pathogens in AD brains.
Therefore, we investigated whether RV might overcome cytotoxic effects of NO excess on septal cholinergic neuroblastoma
SN56 cells. Chronic, 16 h exposure of nondifferentiated SN56
cells (NC) to 0.2 mM sodium nitroprusside (SNP) caused 30
and 27% suppression of pyruvate dehydrogenase (PDH) activity and acetyl-CoA level and the increase of nonviable cells
fraction to 30%. In cAMP/retinoic acid-differentiated cells
(DC), similar 30% inhibition of PDH by SNP caused 50% suppression of acetyl-CoA content and 47% loss of cell viability.
RV (0.005 mM) itself affected none of parameters, neither in
NC nor in DC. It did not overcome SNP-evoked inhibition of
PDH and suppression of acetyl-CoA content in NC and DC.
Despite of that RV partially prevented SNP-induced increase
of DC and NC mortality to 31 and 14%, respectively. These
data demonstrate that greater cytotoxic effects of SNP in DC
than in NC correlated with evoked acetyl-CoA deficits that
might be linked with higher level of acetylcholine metabolism
in the former. Protective effects of RV in NO-challenged cholinergic neurons were not connected with improvement of their
acetyl-CoA metabolism. Supported by MNiSW projects NN401
2333 33, P05A 11030.
342
TVI.07
TRANSMEMBRANE INTRANEURONAL DELIVERY
OF TAU PROTEIN IN RAT MAY SERVE AS AN ANIMAL
MODEL OF HUMAN TAUOPATHY
Mietelska-Porowska A.1, Koss D.2, Baksalerska-Pazera M.1,
Robakiewicz I.1, Nosecka E.1, Riedel G.2, Niewiadomska G.1
1
Department of Neurophysiology, Nencki Insitute of Experimental
Biology PAS, Warszawa, Poland; 2 Department of Life Science of
Medicine, Institute of Medical Sciences, University of Aberdeen,
Aberdeen, UK
Filamentous inclusions of tau protein are hallmarks of tauopathies
including Alzheimer’s disease. Here a rat model for tauopathies
was developed using pore-forming halitoxin Poly-APS. Tau protein was delivered through the membrane into the neurons where
it is metabolized. Rats were injected with recombinant human
Tau441 only (controls), Tau+Poly-APS (double) and Tau+PolyAPS+Okadaic Acid (triple). Cognitive and neurodegenerative
changes were examined with Morrris water maze behavioral test
and immunohistochemistry, respectively. Acquisition of the spatial reference memory was unaffected by administration of Tau
or Tau+Poly-APS. However, enhanced phosphorylation of exogenously delivered tau with the triple infusion impaired learning.
Hippocampal tau was visualized using antibodies against Tau441
and phosphorylated Tau-S404. The higher intensity of Tau441 immunostaining in double and triple groups suggests that exogenous
tau is sequestered from the infusion solution into the cells. With
respect to phospho-tau izoform we observed different cellular
compartmentalization of P-Tau-S404 in double and triple treated
rats. In double treated rats P-TauS401 was distributed both in the
cell bodies and neuronal fibers, while in triple group presence of
OA caused redistribution of phospho-tau staining from neuronal
processes to the perikaryon. This is similar to age-dependent tau
redistribution between cellular compartments and could be a result of hyperphosphorylation.
TVI.08
PAIRED ASSOCIATES LEARNING IN ALZHEIMER’S
DISEASE, MILD COGNITIVE IMPAIRMENT AND
HEALTHY INDIVIDUALS: A PRELIMINARY STUDY
Medygral J.1, Barcikowska M.2, Chodakowska-Zebrowska M.2,
Mandecka M.2, Barczak A.2, Szelag E.1
1
Biology PAS, Warszawa, Poland; 2 Department of Neurology, Central
Clinical Hospital for the Ministry of Internal Affairs
and Administration, Warszawa, Poland
CANTAB Paired Associates Learning test (PAL) involves the
ability to associate the stimulus (what) with its spatial location
(where).There is some evidence that structures of the medial temporal lobe plays a crucial role in formation of these associations.
Evidences from both lesion (in rats, monkeys, humans) and neuroimaging studies suggest that medial temporal lobes structures
such as hippocampus, parahippocampal gyrus and enthorinal cortex are affected in early stages of Alzheimer’s disease (AD). These
stages can be detected using PAL which is a vulnerable tool for
early preclinical detection and assessment of AD. We compared
the performance of PAL in 5 patient with AD, 20 Mild Cognitive
Impairment patients (MCI) and 19 normal healthy controls (NC).
The performance in AD group was significantly lower than those
from MCI and NC group. The main difference was in commit-
ted errors at more difficult, 6-pattern stage, where spatial location
of 6 patterns had to be remembered. Moreover, in this situation
the learning of pattern location in AD group was slowed down;
more trials were needed to complete 6- patterns stage. In summary, these results confirmed that PAL may be the sensitive tool in
clinical diagnosis of AD. Supported by MIND no. WKP_1/1.4.3.1
/2004/11/11/52/2005/U.
TVI.09
MITOCHONDRIAL AND NUCLEAR TARGETS
OF AMYLOID β-EVOKED OXIDATIVE STRESS
Jesko H.1, Strosznajder J.1, Cieslik M.1, Cakala M.1,
Strosznajder R.2
1
Department of Cellular Signalling, 2 Department of Neurosurgery,
Mossakowski Medical Research Centre PAS, Warszawa, Poland.
Amyloid β (Aβ) is responsible for mitochondrial failure and biochemical alterations linked to Alzheimer`s disease (AD). To better
understand mechanisms of Aβ toxicity we investigated its mitochondrial and nuclear targets, apoptosis-inducing factor (AIF) and
Poly(ADP-ribose) polymerase-1 (PARP-1) in PC12 cells transfected with wild type (APPwt) or double Swedish-mutated human
Amyloid Precursor Protein gene (APPsw) characterized by different Aβ concentrations. We found close relationship between Aβ
level and cyclooxygenase (COX)- and lipoxygenase (LOX)-related
free radical formation leading to p65/NF-κB nuclear translocation. COX and LOX inhibitors protected APPsw cells against p65
translocation. Aβ-evoked oxidative stress enhanced mitochondrial
AIF level and inhibited PARP-1 in APPsw cells. Nitrosative stress
evoked by 0.5 mM sodium nitroprusside (SNP) had no further effect on Aβ-altered PARP-1 activity and mitochondrial AIF level
in APPsw cells. However, SNP evoked death of 70–80% of all
cell types after 24 h. COX and LOX inhibitors had ameliorating
effect in these conditions. Our data indicated that double Swedish mutation in APP significantly increased cell vulnerability to
oxidative stress. Enhanced mitochondrial AIF level and PARP-1
inhibition might be responsible for cell survival under oxidative
stress evoked by accumulating Aβ in APPsw cells. COX and LOX
inhibitors protected the cells against death caused by simultaneous
Aβ toxicity and nitrosative stress.
TVI.10
PRIMING OF ROTATIONAL BEHAVIOR
BY A DOPAMINE RECEPTOR AGONIST IN
HEMIPARKINSONIAN RATS:
MOVEMENT-DEPENDENT INDUCTION
Simola N.1, Di Chiara G.1, Daniels W.2, Schallert T.3, Morelli M.1
1
Department of Toxicology, University of Cagliari, Cagliari, Italy; 2
Discipline of Human Physiology, University of
KwaZulu-Natal, Westville Campus, Durban, South Africa; 3
Department of Psychology, University of Texas, Austin, TX, USA
Repetitive stimulation of basal ganglia dopamine receptors leads to
abnormal motor responses in dopamine-denervated rats. To study
whether such responses were influenced by the previous execution
of movement, we evaluated how “priming”, a phenomenon mimicking an abnormal motor response, depends on movement performance. To this end, unilaterally 6-hydroxydopamine-lesioned
rats received apomorphine (0.2 mg/kg s.c.), being either allowed to
move or immobilized (1 h) before, concomitantly to, or after its administration. Three days later, the dopamine D1 receptor agonist
SKF 38393 (3 mg/kg s.c.) was administered. Rats that had rotated
following apomorphine showed contraversive rotational behavior
following SKF 38393, whilst rats that had been immobilized concomitantly to apomorphine, but neither before nor after, did not.
To clarify whether immobiliztion-related stress influenced the results, additional rats received apomorphine plus immobilization
and the corticosterone-synthesis inhibitor metyrapone (100 mg/
kg i.p.), or apomorphine plus a tail stressor, being not immobilized. Metyrapone did not affect the prevention of priming by immobilization, and tail stressor did not affect priming magnitude,
suggesting that stress has minimal effect on the results observed.
This study demonstrates how movement performance following
initial dopaminergic stimulation governs the occurrence of an abnormal motor response to a subsequent dopaminergic challenge in
dopamine-denervated rats.
TVI.11
STUDY OF THE ROLE OF ALPHA-SYNUCLEIN
PHOSPHORYLATION AT Ser 129 IN DOPAMINERGIC
NEURODEGENERATION IN PARKINSON’S DISEASE
Febbraro F.1, Jensen P.1, Kirik D.2, Romero-Ramos M.1
1
Department of Medical Biochemistry, Aarhus University, Aarhaus,
Denmark; 2 B.R.A.I.N.S., Department of Experimental Medical
Science, Lund University, Lund, Sweden
Studies during the last decade have established a central role in
Parkinson’s disease (PD) neurodegeneration for the protein alphasynuclein (α-syn). Among the different post-translational modifications that human a-syn can undergo, phosphorylation has been
related to the disease. α-syn phosphorylation at Ser 129 seems
to be related to the disease progress in PD, since S129P-α-syn is
found in low levels in healthy humans and it is related to increased
inclusion formation. In order to elucidate the role of phosphorylation of α-syn at Ser 129 in the disease process we used a rat PD
model based on the local injection of recombinant adeno-associated viral vectors (rAAV). We created mutations in α-syn at Ser129,
replacing the serine either with alanine (S129A) to block phosphorylation or with aspartate (S129D) to mimic phosphorylation. We
overexpressed wt, S129A and S129D α-syn in rat midbrain and
thereafter have compared the effect of the overexpression on dopaminergic neurons at different time points post-injection. Our results show that pseudophosphorylated α-syn S129D induced faster
motor deficit compared to S129A. On our hands, the phosphorylation of α-syn at Ser129 seems to have a role in the progression of
the disease but is not necessary for pathology, motor deficits or cell
death to occur.
TVI.12
CHRONIC, INTRAVENTRICULAR INFUSION
OF MPP+ WITH ALZET OSMOTIC MINIPUMPS IN
RATS AS A MODEL OF PARKINSON’S DISEASE
Kuter K.1, Kolasiewicz W.1, Zapala M.1, Kowalska M.2,
Golembiowska K.2, Morelli M.3, Wardas J.1
1
Department of Neuropsychopharmacology, 2 Department of
Pharmacology, Institute of Pharmacology PAS, Kraków, Poland;
3
Department of Toxicology, University of Cagliari, Cagliari, Italy
An underlying mechanism of degeneration in Parkinson’s disease (PD) is unknown. The animal models of PD, developed
so far have certain disadvantages; hence a search for a new
model of PD seems necessary. Chronic, unilateral, intraventricular delivery of MPP+ (0.284 and 0.428 mg/kg/day for 28
days) using an ALZET osmotic minipump, implanted s.c.,
produced a marked, dose-dependent loss of DA and its metabolites DOPAC and HVA (50–90%) in the striatum, ipsilateral to the infusion site. DA concentration was normal in the
non-infused, right striatum. Also no changes in the 5-HT level
were observed. The stereological counting of the number of
dopaminergic neurons in the substantia nigra pars compacta
(SNc), stained with the antibody against tyrosine hydroxylase,
showed their 30–50% loss on the lesioned side. Those changes
were accompanied with a diminished expression of mRNA
for the dopamine transporter in the SNc (by ca. 30%). Additionally, in situ hybridization studies indicated an enhanced
expression of mRNA for both adenosine A 2A and dopamine D2
receptors in the striatum and diminished expression of mRNA
for BDNF in the hippocampus. The obtained results showed
that this chronic model of continuous, intracerebral infusion of MPP+, produced a selective nigrostriatal DA cell loss
and number of other neurochemical changes resembling PD.
Study supported by the grant No. NN401 1137 33 (MS&HE)
and by a statutory fund from the Institute of Pharmacology,
PAS, Poland.
TVI.13
A 2A ADENOSINE RECEPTOR ANTAGONISTS EFFECT
ON HYDROXYL RADICAL GENERATION IN DA
DEPLETED STRIATUM WITH RESERPINE
Golembiowska K.1, Dziubina A.1, Morelli M.2
1
Department of Pharmacology, Institute of Pharmacology PAS,
Kraków, Poland; 2 Department of Toxicilogy, University of
Cagliari, Cagliari, Italy
Although the cause of DA neurons neurodegeneration is still
unknown, oxidative stress is paramount in the pathogenesis
of Parkinson’s disease. An accumulation of cytosolic DA has
been shown to be neurotoxic through the generation of free
radicals (FR). Searching for FR scavengers, we studied the effect of selective A 2A adenosine receptor antagonists, shown to
have neuroprotective properties, on hydroxyl radical (HR) production in rat striatum with reserpine impaired DA storage. We
found an increase in extracellular glutamate and HR levels in
DA-depleted striatum. CSC (1 mg/kg), ZM 241385 (3 mg/ kg)
and L-DOPA (25 mg/kg) normalized glutamate release and
combination of A 2A antagonists and L-DOPA showed similar
effect. CSC increased DA and HR levels but ZM 241385 given
alone did not affect DA nor HR levels. L-DOPA enhanced DA
extracellular level but did not change the production of HR.
Combination of L-DOPA and CSC further elevated DA extracellular level and markedly increased HR production while
combination of L-DOPA and ZM 241385 attenuated, enhanced
by L-DOPA DA level and had no effect on HR production.
This data suggests that disrupted balance between DA and
glutamate in DA depleted nigrostriatal neurons results in generation of neurotoxic HR. Both A 2A antagonists, like L-DOPA,
redress the DA/glutamate balance. However, A 2A antagonists
in combination with L-DOPA show different pharmacological
profile in their effect on DA release and subsequent generation
of HR.
344
TVI.14
HIGH FREQUENCY STIMULATION OF THE
SUBTHALAMIC NUCLEUS INCREASES BLOOD
NK CELL NUMBER IN HEMIPARKINSONIAN RATS
DIFFERING IN RESPONSE TO NOVELTY
Grembecka B., Glac W., Plucinska K., Ledochowski P.,
Gora A., Badtke P., Wrona D.
Department of Animal Physiology, Gdansk University, Gdańsk,
Poland
High frequency stimulation (HFS) of the subthalamic nucleus (STN)
is a surgical therapy for improving of motor symptoms in Parkinson’s disease. In this study, we assessed the HFS effects on cytometric analysis of the peripheral blood lymphocytes (T, B, NK, T
helper, T cytotoxic) in freely moving hemiparkinsonian rats. Before
unilateral lesion of the right substantia nigra pars compacta (6-hydroxydopamine), all rats were divided into two behavioral groups:
high-HR and low-LR responders in novelty test. As compared to the
sham controls, HFS of STN significantly increases NK cell percentage number (25.86 ± 9.68% vs. 18.79 ± 5.63%, P<0.05). In contrast,
significantly (P<0.05) lower B lymphocytes level in stimulated (18.86
± 3.91%) then sham group (22.80 ± 4.63%) was observed. These general changes in NK and B lymphocyte numbers were reflected in HR
stimulated animals (31.08 ± 7.19% vs. 20.44 ± 5.65% for NK cells
number and 17.67 ± 2.57% vs. 23.23 ± 3.37% for B lymphocyte, stimulated vs controls, respectively, P<0.01). On the other hand, significantly lower T lymphocyte level in LR stimulated animals in comparison to LR controls was found (38.32 ± 4.18% vs. 45.91 ± 5.68%).
Moreover, significant differences between stimulated HRS and LRS
in the NK and T cytotoxic percentages were found (31.08 ± 7.19%
vs. 17.63 ± 6.74%, P<0.05; 8.46 ± 1.83% vs. 12.29 ± 1.76%, P<0.01).
The results emphasize the importance of individual differences in
reactivity to novelty on immune response to HFS of STN.
TVI.15
AN INVOLVEMENT OF MAPK/ERK1/2 PATHWAY IN
MECHANISM OF LACTACYSTIN-INDUCED CELL
DAMAGE IN PRIMARY CORTICAL NEURONS
Jantas D.1, Konieczny J.2, Lenda T.2, Lason W.1, Lorenc-Koci E.2
1
Department of Experimental Neuroendocrinology, 2 Department of
Neuropsychopharmacology, Institute of Pharmacology PAS, Kraków,
Poland
Proteasome dysfunction is involved in pathomechanism of several
neurodegenerative diseases where an accumulation of aberrant
proteins occurs (e.g. Parkinson’s disease, Alzheimer’s disease).
Lactacystin (LC) has been used for induction of proteasome inhibition-dependent neuronal cell death for several years but mechanism of its toxic action on neurons is still poorly understood. In the
present study we showed time- and concentration-dependent toxic
action of lactacystin (0.25–50 μM) in mouse cortical neurons. Although, lactacystin induced caspase-3 activation, its toxic action
was not attenuated by caspase-3 inhibitor AcDEVD-CHO. We
demonstrated that inhibitors of MAPK/ERK1/2 cellular signaling (U0126 and PD98052) were protective against LC-evoked cell
death as confirmed by LDH and MTT reduction assays. Moreover,
these data were verified by Western Blot analysis, where we observed the increase in ERK1/2 activity after LC treatment and this
effect was inhibited by U0126. The obtained data point to engagement of activation of MAPK/ERK1/2 in toxic action of lactacystin
and give a rationale for using agents which inhibit this intracellu-
lar pathway in treatment of neurodegenerative diseases connected
with proteasome dysfunction. Supported by Polish MNSW Scientific Network Fund no 26/E-40/BWSN-0023/2008.
TVI.16
PARKINSON’S DISEASE LEADS TO DISTORTED
INTERACTION OF EPISODIC AND WORKING
MEMORY SYSTEMS
Gawrys L.1, Szatkowska I.1, Pilacinski A.1,
Piatkowska-Janko E.2, Bogorodzki P.2, Wolak T.3, Friedman A.4,
Sienkiewicz J.4, Koziorowski D.4, Janik P.4, Jamrozik Z.4,
Krolicki L.4, Andrysiak R.4, Kulinski R.4, Kaczmarek L.1
1
Nencki Institute of Experimental Biology PAS, Warszawa, Poland;
2
Warsaw University of Technology, Institute of Radioelectronics,
Warszawa, Poland; 3 Institute of Physiology and Pathology of Hearing,
Warszawa, Poland; 4 Medical University of Warsaw, Warszawa,
Poland
Patients with Parkinson’s disease (PD) exhibit impairments in executive function and working memory. As opposed to motor symptoms of the disease the physiological origins of cognitive deficits
are not well understood. The aim of the present study was to delineate mechanisms of cognitive impairment in PD using blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI). Twenty-two patient and 10 matched control subjects
accomplished Wisconsin Card Sorting Test (WCST) – standard
clinical measure of executive function and then underwent fMRI
during performance of the N-Back working memory task.The correlation analysis comparing BOLD signal intensity changes with
number of categories achieved in WCST revealed a significant
positive correlation within supramarginal gyrus, inferior parietal
gyrus, middle and inferior frontal gyrus and caudate nucleus.
Significant negative correlations with executive impairment were
observed within fusiform and parahippocampal gyrus, as well as
enthorhinal and perirhinal cortex. These results suggest that cognitive impairment in PD may origin in consequence of distorted
interaction between episodic and working memory systems.
TVI.17
INHIBITORY EFFECTS OF 17β-ESTRADIOL ON
PRODUCTION OF BOTH MOLECULAR AND
CELLULAR FACTORS IN NEUROINFLAMMATORY
REACTION IN MODEL OF PARKINSON’S DISEASE
Ciesielska A.1,2, Joniec I.2, Cudna A.2, Kurkowska-Jastrzebska I.1,2,
Sznejder A.2, Zaremba M.2, Czlonkowska A.1,2, Czlonkowski A.2
1
Second Department of Neurology, Institute of Psychiatry and
Neurology, Warsaw, Warszawa, Poland; 2 Departament of Clinical
and Experimental Pharmacology, Medical University of Warsaw,
Warszawa, Poland
The neuroprotective action of 17β-estradiol (E2) against 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown in
both female and male mice, however the exact mechanisms of that
phenomenon remain obscured. We studied the chronic effects of
E2 (0.25 mg per pellet, 21-days release) administered 7 days prior
(Experiment 1) to or 3 days after (Experiment 2) MPTP intoxication (40 mg/kg, i.p.) in C57BL aged male mice on neurodegenerative and inflammatory processes in nigrostriatal pathway. We
estimated striatal: tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) content (Western blotting); cytokines (TNFα,
TGFβ1, IFNγ), trophic factor (GDNF) gene expression (RT-PCR);
CD4+ and CD8+ cells influx (immunohistochemistry) at 1, 7, 21
(Exp. 1) and 7, 21 (Exp. 2) days post intoxication. E2 exerted a
neuroprotective effect upon nigrostriatal system when administered prior but also when administered after intoxication (E2 attenuated the MPTP-induced loss of TH). E2 also decreased the
GFAP content. MPTP caused a rapid increase of TGFβ1, TNFα
and IFNγ. Pre-treatment with E2 decreased the early expression
of TGFβ1 and IFNγ but failed to suppress the MPTP-induced increase of TNFα. E2 pre-treatment also induced an increase of the
GDNF and CD4+ cells influx to the injured brain areas but decreased the CD8+ cells infiltration. The neuroprotective effects of
E2 indicated in MPTP model might mediate through a modulation
of neuroinflammatory reaction in lesioned nigrostriatal system.
TVI.18
THE MOLECULAR AND BEHAVIORAL CHANGES IN
THE LONG-TERM OBSERVATIONS IN MICE MODEL
OF PARKINSON’S DISEASE
Zaremba M.1, Joniec I.1, Piechal A.1, Pyrzanowska J.1,
Kurkowska-Jastrzebska I.2, Ciesielska A.1,2, Czlonkowska A.1,2,
Czlonkowski A.1
1
Department of Experimental and Clinical Pharmacology, Medical
University of Warsaw, Warszawa, Poland; 2 2nd Department of
Neurology, Institute of Psychiatry and Neurology, Warszawa, Poland
The pathogenesis of non-motor symptoms in Parkinson’s disease
(PD) is complex and not fully understood, but is believed to be
related to the widespread nature of dopaminergic dysfunction involving structures beyond the substantia nigra. Therefore we’ve
also focused in our experiments on specific brain regions (e.g.
hippocampus and prefrontal cortex) critically involved in spatial
learning and memory processes. One year old C57/BL male mice
received 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP)
40 mg/kg, and control group received 0.9 % NaCl. To evaluate spatial learning and memory abilities, the Morris water maze (WM)
behavioral test was provided after 6 months from the intoxication.
The brain concentration of monoamines: dopamine (DA), noradrenaline (NA), 5 – hydroxytryptamine (5-HT) and amino acid:
glutamate (Glu) were determined by using high – performance
liquid chromatography (HPLC). Statistical significances differences in monoamines levels between groups were noticed in hippocampus (NA, DA content) and prefrontal cortex (NA content).
We didn’t notice significant differences in the WM test parameters
between MPTP and the control animals. Correlation between the
results of the behavioral testing in the probe trial and the level of
monoamines were calculated. Swim distance to reach the hidden
platform was negatively correlated with NA level in the hippocampus (rp=−0.62, P<0.05). There is need for a further investigation to
confirm the role for NA in spatial memories.
TVI.19
NEUROPROTECTIVE EFFECTS OF AAV2-hIL-10
IN MOUSE MODEL OF PARKINSON`S DISEASE
Joniec I.1, Ciesielska A.1,2, Gladka A.1, Cudna A.1, Zaremba M.1,
Kurkowska-Jastrzebska I.2, Hadaczek P.3, Bankiewicz K.3,
Czlonkowska A.1,2, Czlonkowski A.1
1
Department of Experimental and Clinical Pharmacology, Medical
University, Warszawa, Poland; 2 Second Department of Neurology,
Institute of Psychiatry and Neurology, Warszawa, Poland;
3
Department of Neurosurgery, University of California San
Francisco, San Francisco, CA, USA
Parkinson’s disease (PD) is a neurodegenerative disorder which
is characterized by abnormal loss of nigrostriatal dopamine (DA)
neurons, accompanied by DA deficiency in the striatum. The pathomechanism by which DA neurons degenerate is still unknown,
however, there is increasing evidence that is possible immunological mechanisms involvement in the etiopathogenesis of PD. The aim
of the present study was to examine the effect of an adeno-associated viral type-2 (AAV2) vector containing human interleukin-10
(hIL-10) gene on dopaminergic system restoration in the murine
model of PD induced by by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL/6 mice 12 months-old were used
in this study. MPTP was injected in four intraperitoneal injections
at 1-h intervals. AAV2-hIL-10 vector was bilaterally administered
into striatum at 14, 21 or 28 days prior to MPTP intoxication. Animals were sacrificed at 7 days following MPTP injection. Striatal
DA, DOPAC, HVA concentrations were quantified by HPLC method; tyrosine hydroxylase (TH) mRNA expression was measured
by RT-PCR method. MPTP treatment dramatically decreased DA
concentration, significantly decreased TH mRNA gene expression.
AAV2-hIL-10 exerted a neuroprotective effect upon dopaminergic
system (lower decrease in DA concentration). Additionally, viral
vector administration prevented depletion of TH mRNA expression induced by MPTP. Our data suggest that AAV2-hIL-10 may
play a neuroprotective role in MPTP mouse model of PD.
TVI.20
EFFECTS OF AAV2-hIL-10 ON IMMUNE
RESPONSE FOLLOWING TOXIC DEGENERATION
CAUSED BY 1-METHYL-4-PHENYL-1,2,3,6TETRAHYDROPYRIDINE IN MICE
Joniec I.1, Ciesielska A.1,2, Gladka A.1, Schwenkgrub J.1,
Sznejder A.1, Cudna A.1, Hadaczek P.3, Bankiewicz K.3,
Czlonkowska A.1,2, Czlonkowski A.1
1
Medical University, Warszawa, Poland; 2 Second Department of
NeurologyInstitute of Psychiatry and Neurology, Warszawa, Poland;
3
Department of Neurosurgery, University of California San
Francisco, San Francisco, CA, USA
Interleukine 10 (IL-10) – an antiinflammatory cytokine produced by
lymphocytes and mononuclear phagocytes including microglia. IL10 modulates the biological activities of immune cells resulting in a
decreased production of pro-inflammatory mediators including cytokines, chemokines and adhesion molecules. The aim of the present
study was to examine the effect of an adeno-associated viral type-2
(AAV2) vector containing human interleukin-10 (hIL-10) gene to
evaluation of immune response to the AAV2-hIL-10 (measured as
IFN-γ, GFAP and TGFβ mRNA expression) in the murine model of
Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL/6 mice 12 months-old were
used in this study. MPTP (40 mg/kg) was injected in four intraperitoneal injections at 1-h intervals. AAV2-hIL-10 vector was bilaterally administered into striatum at 14, 21 or 28 days prior to MPTP
intoxication. Animals were sacrificed by spinal cords dislocation at
7 days following MPTP injection. TGFβ, IFN-γ and GFAP mRNA
expression was examined by RT-PCR method. MPTP treatment
significantly increased IFN-γ mRNA expression. AAV2-hIL-10 administration strongly increased IFN-γ as well as TGFβ and GFAP
(21 and 28 day) gene expression compared to control and MPTP
group. Our results point to the necessity of the reinterpretation of the
role of the inflammatory reaction in nerodegenerative processes
346
TVI.21
THE SLEEP PROFILE IN PARKINSON’S DISAEASE
Antczak J.1, Rakowicz M.1, Derejko M.1, Zalewska U.1,
Sienkiewicz J.2, Banach M.1, Tarnacka B.3, Jernajczyk W.1
1
Department of Clinical Neurophysiology, Institute of Psychiatry and
Neurology, Warszawa, Poland; 2 Department of Neurology, Warsaw
Medical University, Faculty of Health Science, Warszawa, Poland;
3
Second Department of Neurology, Institute of Psychiatry and
Neurology, Warszawa, Poland
Sleep disorders are reported by up to 90% of the Parkinson’s disease (PD) patients. The main causes of poor sleep quality are the
altered dopaminergic transmission in the subcortical areas, playing role in the sleep wake regulation, the influence of dopaminergic medication and the negative effect of rigidity, painfull dystonias, and other motor symptoms of PD. The aim of this study
was the objective polysomnographic assessment of sleep quality
in PD with respect to the possible gender differences. We evaluated: seven female and 10 male PD patients (mean age 66.3 ± 9.2
vs. 63.9 ± 10.2 years; mean Hoehn Yahr stage 2.2 ± 0.5 vs. 2.3
± 0.5 and mean UPDRS score 28.0 ± 9.3 vs. 33.9 ± 15.9) respectively, with no significant differences between both sexes. The
sleep was examined with nocturnal polysomnography and with
two questionnaires – the Pitsburger sleep quality index (PSQI) and
the Parkinson disease sleep scale (PDSS). Both genders showed
worsened sleep quality. In particular the decreased sleep efficiency (61.1 ± 17.5% women, 45.5 ± 23.9% men) and prolonged sleep
onset latency (95.2 ± 101.6 min – women, 52.6 ± 77.7 min – men)
showed marked differences with respect to estabilished norms for
the Polish population. The statistical comparison of the polysomnographic parameters revealed no significant differences between
both genders. The poor sleep quality seen in our polysomnographic findings stays in accordance with frequent complainings of disordered sleep among PD patients.
TVI.22
INHIBITION OF INFILTRATION AND ACTIVATION
OF BRAIN MACROPHAGES UNDERLIES ANTITUMOR
ACTIVITY OF SYSTEMICALLY ADMINISTERED
CYCLOSPORIN A
Gabrusiewicz K., Ellert-Miklaszewska A., Sielska M.,
Frankowska M., Kaminska B.
Malignant glioblastomas are characterized by infiltration of tumour tissue with brain macrophages that contribute to tumour progression through release a variety of growth factors, cytokines/
chemokines supporting tumor growth, invasion and the immune
system evasion. Thus targeting of cytokine production, infiltration
and activation of macrophages may be a novel antitumor strategy.
In the present study, we investigated an effectiveness and molecular mechanisms mediating antitumor effects of CsA in the murine
glioma model. EGFP-GL261 glioma cells were injected into the
striatum of C57BL/6 mice and tumor-bearing mice received CsA
(2 or 10 mg/kg/i.p.) every 2 days from 2nd or 8th day after implantation. CsA-treated mice had significantly smaller tumors than
control mice. When the treatment was postponed to 8th day, only
the higher dose of CsA was effective. CsA-treated mice showed
a diminished number of tumor-infiltrating, amoeboid brain macrophages. TUNEL staining revealed a DNA fragmentation mostly
within infiltrating macrophages after CsA treatment. At concentrations <10 μM CsA did not affect proliferation or viability of cultured glioma cells. Elevated IL-10 and GM-CSF levels were found
in tumor-bearing hemisphere in comparison to naive controls and
CsA treatment reduced significantly cytokine levels. Our findings
demonstrate that CsA-induced decrease of cytokine production,
infiltration and activation of macrophages results in reduction of
glioma growth and progression.
TVI.23
EVALUATION OF MIGRATION OF BONE MARROWDERIVED MACROPHAGES AND LYMPHOCYTES
TOWARDS GLIOMA CELLS AND AND ITS
MODULATION BY CYCLOSPORIN A
Sielska M.1, Gabrusiewicz K.1, Kwiatkowska E.2, Kowalczyk D.2,
Ellert-Miklaszewska A.1, Kaminska B.1
1
Biology PAS, Warszawa, Poland; 2 Department of Cancer Diagnostics
and Immunology, The Great Poland Cancer Center, Poznań, Poland
Malignant glioblastomas are characterized by infiltration of tumour tissue with brain macrophages that may consist up to 30%
of tumour mass and contribute to tumour progression. A relative
contribution of resident microglia and peripheral monocyte/macrophages in gliomas is poorly defined. We generated chimeric
mice with the immune system reconstituted after irradiation with
hematopoietic GFP-bone marrow cells. The dsRed-GL261 glioma
cells were implanted to the brains of 16-weeks old C57BL/6 chimeric mice Two weeks after implantation, tumour bearing hemispheres were isolated and the number of CD11b+CD45low microglial cells or CD11b+CD45high macrophages was determined by flow
cytometry. We found that peripheral GFP+ macrophages comprise
above 60% of GFP+ cells in the tumor. A co-localization of Iba-1+
cells (macrophages/microglia) with GFP+ cells has been detected
by confocal microscopy. Tumor associated peripheral macrophages can facilitate glioma invasion and promote angiogenesis. We
have previously demonstrated that cyclosporin A (CsA) blocks activation of microglia and its promoting effects on glioma invasion
in vitro and in vivo (Sliwa et al. 2007). In chimeric mice treated
CsA, a percentage of GFP+ macrophages in gliomas was reduced
suggesting an inhibitory effects of CsA on immune cell migration.
Our studies demonstrate that blood-borne macrophages migrate to
the tumour and consist a significant population of tumour-associated macrophages.
TVI.24
CELL SIZE AND PROLIFERATION OF
SUBEPENDYMAL GIANT CELL ASTROCYTOMAS
ARE REGULATED BY mTOR AND Ras-ERK SIGNALING
PATHWAYS
Tyburczy M.1, Kotulska K.2, Jozwiak S.2, Kaminska B.1
1
Biology PAS, Warszawa, Poland; 2 Department of Pediatric
Neurology, The Children’s Memorial Health Institute, Warszawa,
Poland
Tuberous sclerosis complex (TSC) is characterized by cortical developmental malformations that are associated with epilepsy and
appearance of benign brain tumors – subependymal giant cell astrocytomas (SEGAs) which are composed of distinct cell types,
including giant cells and dysplastic neurons. TSC results from
mutations in TSC1 and TSC2, which lead to the mTOR pathway
activation, and p70S6 kinase and ribosomal S6 protein phosphorylation. ERK pathway is also aberrantly activated in SEGAs. Clinical trials with the mTOR inhibitor – rapamycin have demonstrated
reduction in size of SEGAs, but the molecular mechanisms are unknown. In the present study, we evaluated the effects of rapamycin
and the ERK pathway inhibitor – UO126 on cell size, proliferation
and viability of cell cultures derived from SEGA. Rapamycin or
UO126 alone did not affect viability and proliferation of SEGA
cells. However, treatment with both drugs reduced proliferation
of SEGA cells. Staining of F-actin revealed decrease of cell size
in SEGA cultures exposed to rapamycin alone or in combination
with UO126, but treatment with UO126 alone did not influence cell
size nor morphology. Our studies demonstrate that simultaneous
inhibition of both mTOR and ERK signaling pathways reduces
proliferation of SEGA cells. Moreover, inhibition of mTOR signaling with rapamycin diminishes a volume of giant SEGA cells.
It implicates that inhibitors of mTOR and ERK pathways should
be considered for clinical trials of SEGAs
TVI.25
DIFFERENTIAL MODULATION OF BASAL
AND IFN-INDUCED STAT TARGET GENES
EXPRESSION BY SPECIFIC STAT DIRECTED
OLIGODEOXYNUCLEOTIDE DECOYS
IN GLIOMA C6 CELLS
Adach-Kilon A., Dabrowski M., Kaminska B.
Constitutive activation of STAT3, leading to up-regulation of some
proliferation-related and anti-apoptotic genes, was described in a
number of tumors, including glioblastomas and is considered as
an important drug target for cancer therapy. The binding sites for
STAT3 are highly similar to that of STAT1, and the two factors
may have opposite effects on expression of the same target genes.
We took advantage of differences in binding sequences, relatively specific for some but not all STAT proteins, to elucidate the
interplay between different STATs in the regulation of gene expression in the rat glioma C6 cells. A number of double-stranded
oligodeoxynucleotide (ODN) decoys, each carrying a particular
STAT binding sequence, were employed as competitive inhibitors
of binding sites in the genome. We compared the effects of the decoy containing ISRE/GAS (binding complexes of STAT1, STAT2,
IRFs) or STAT1 site (specific for STAT1 homodimer) on the expression of endogenous genes. We report that the decoy against
STAT1 reduced the constitutive expression of endogenous STAT3
target genes under basal conditions. The decoy ISRE/GAS blocked
IFNg-induced increase in expression of STAT target genes. Decoys with mutated and/or scrambled STAT-binding motifs were
used as controls for specificity. Our results demonstrate a limited
usefulness of some ODN decoys in manipulation of endogenous
gene expression, probably due to limited sequence specificity of
STAT binding sites.
TVI.26
CRITICAL PERIOD FOR MOTION PERCEPTION
IN CATS
Zapasnik M., Kossut M., Burnat K.
Visual input guides the development of visual system in which
distinct critical periods are connected with separate functions. Monocular deprivation during critical period leads to
acuity deficits, while binocular deprivation impairs more specific visual functions. Children with later onset of binocular
cataract have normal global motion perception, while patients
with congenital cataract show a deficit (Lewis and Maurer
2005). Similarly global motion detection is impaired in cats
binocularly deprived for 6 months (Burnat et al. 2002). The
exact time-frame for global motion perception critical period
still needs to be established. Two experimental groups of cats:
congenital (2BD) binocularly deprived for fi rst 2 months of
life and developmental (2N2BD) with 2 months of normal vision followed by 2 months of deprivation were used. Controls
had normal visual input. Animals were trained in two-choice
apparatus for a food reward. Visual discrimination tasks with
random dot patterns differing in velocity and, or direction
were used. In 2N2BD group we found significant impairment as compared to 2BD group in velocity discrimination
tasks. Moreover, we found in 2N2BD group that the detection of global motion signal at low contrast is impaired by
the addition of dark noise. Surprisingly, visual experience for
fi rst 2 months limited to the moving shadows may facilitate
global motion perception as the 2BD group learned motion
tasks faster than control group. Supported by MNiSW grant
N40132/1002.
TVI.27
A MIFEPRISTONE-REGULATED ADENO-ASSOCIATED
VIRAL VECTOR SYSTEM FOR REGULATED
NEUROTROPHIC FACTOR EXPRESSION
IN THE CENTRAL NERVOUS SYSTEM
Maddalena A., Tereschchenko Y., Bähr M., Kügler S.
Department of Neurology, University Hospital of Goettingen,
Goettingen, Germany
Neurotrophic factors (NF) are potent molecules with great
promise for treatment of devastating neurodegenerative disorders like Alzheimer´s and Parkinson´s disease. Targeted delivery of NF like NGF or GDNF by means of viral vectors to
distinct populations of brain cells may significantly enhance
bio-availability and safety of such treatment approaches. However, gene therapy in its current understanding means to introduce e.g. a cDNA coding GDNF into neurons or astrocytes and
expressing the factor from that time on continuously, without
an option to tune expression levels according to individual patient’s demands or to shut down expression in case of unforeseen side effects. In order to increase efficacy and safety of
gene therapy vectors for treatments of human patients, we here
describe the development of a tightly regulatable vector system
based on the approved human drug, mifepristone, for applications in the CNS. A two-vector based layout allows modulating levels of the transactivator in order to achieve a very tight
off-state while maintaining sufficient potency for activation of
transgene expression. We describe characterization of the vector system in cultured primary brain cells and in the rat striatum and cortex in terms of dependence of vector titres, inducing drug levels, and repeated responsiveness. We believe that
such vector system will significantly promote gene therapeutic
applications in patients but will also have great impact on basic
research applications.
348
TVI.28
PRESYNAPTIC AND POSTSYNAPTIC EFFECTS OF
NORADRENERGIC FUNCTION IN AN ATTENTIONAL
SET-SHIFTING TEST
Bondi C.1, Jett J.2, Morilak D.2
1
Department of Neuroscience, University of Pittsburgh, Pittsburgh,
PA, USA; 2 Department of Pharmacology, University of Texas Health
Science Center at San Antonio, San Antonio, TX, USA
Chronic stress is a major risk factor for many psychiatric disorders, which are also associated with prefrontal cortical dysfunction. We found that elevating norepinephrine (NE) in rat medial
prefrontal cortex (mPFC) with atipamezole enhanced extra-dimensional (ED) cognitive set-shifting on the attentional set-shifting test
(AST). By contrast, a 2-week exposure to chronic unpredictable
stress (CUS) induced an antidepressant (DMI)-reversible deficit on
ED. To assess whether CUS-induced cognitive deficits represent a
loss of NE modulation in the mPFC, we tested if elevating NE activity in the mPFC is still capable of enhancing cognition after CUS.
Again, CUS impaired ED cognitive set-shifting in saline treated
rats, but this deficit was attenuated in CUS rats given atipamezole
prior to testing. Extracellular NE was not different between salinetreated CUS and control rats. However, atipamezole enhanced
NE release in both control and CUS rats throughout the duration
of the test. In the second experiment, CUS produced a cognitive
deficit, which was prevented by chronic DMI. Acute blockade of α1adrenergic receptors in mPFC of CUS/DMI-treated rats prior to ED
caused a cognitive deficit similar to that of CUS/saline rats. These
results suggest the beneficial effects of chronic antidepressant treatment on cognitive performance are mediated through α1-adrenergic
receptors in the mPFC. Furthermore, NE facilitation of cognitive
flexibility in the mPFC remains intact after CUS.
TVI.29
ELEVATED EXPRESSION OF α1-ADRENOCEPTOR
SUBTYPES IN THE HIPPOCAMPUS OF RATS NONRESPONDING TO IMIPRAMINE TREATMENT
IN THE CHRONIC MILD STRESS
Bielawski A.1, Kowalska M.1, Papp M.2, Nalepa I.1
1
Department of Brain Biochemistry, 2 Laboratory of Behavioral
Pharmacology, Deptartment of Pharmacology, Institute of
Cerebral α1-adrenoceptors (α1-AR) are known to be changed by
stress conditions and involved inthe mechanism of antidepressant action. The chronic mild stress (CMS) procedure that induces depression-like symptoms in animals is a useful tool to study
the mechanisms of action of antidepressant drugs in animals. We
aimed to investigate the expression of α1A-, α1B-, and α1D-AR mRNAs in the hippocampus of rats subjected to the standard CMS
procedure and then treated with an antidepressant drug, imipramine (IMI). Five groups of male Wistar rats were considered in
the study: sham-saline; stress-saline; sham-IMI; stress-IMI and
IMI-non-responders (i.e., stressed rats, which did not respond to
IMI treatment). The expression of α1-ARs was measured at the
level of mRNA (by quantitative real-time PCR) and their total
density analyzed by [3H]prazosin autoradiography. We found that
both CMS procedure and IMI-treatment did not affect the expression of all α1-AR mRNAs. However, in rats non-responding to IMI
treatment in behavioral test, the expression of α1A- , α1B- and α1D-AR
mRNAs was significantly increased (respectively, by 81%, P<0.01;
78%, P<0.01; 46%, P<0.5) compared to sham-saline and stressIMI groups. Similar direction of change was seen in α1-AR total
density. Our results suggest the involvement of all subtypes of α1adrenoceptor in the phenomenon of resistance of depressive animal to IMI treatment. Supported by statutory funds of the Institute
of Pharmacology, PAS.
TVI.30
EXAMINATION OF SCHIZOPHRENIA
ENDOPHENOTYPES IN THE HETEROZYGOUS
NEUREGULIN-1 KO MOUSE:
A PSYCHOPHARMACOLOGICAL AND MAGNETIC
RESONANCE IMAGING STUDY
O’Tuathaigh C.1, O’Sullivan G.1, Tighe O.1, O’Leary C.1,
Desbonnet L.1, Lai D.2, Harvey R.2, Croke D.1, Waddington J.1
1
Department of Molecular and Cellular Therapeutics, Royal College
of Surgeons in Ireland, Dublin, Ireland;2 Department of Developmental
Biology, Victor Chang Cardiac Research Institute, Sydney, Australia
Meta-analysis has confirmed the association between the neuregulin-1 (NRG1) gene risk to develop schizophrenia. Neuregulin-1,
alongside several other risk genes, is known to play a role in glutamatergic neurotransmission. There is a substantive body of evidence
for hypoglutamatergic function in schizophrenia; the psychotomimetic activity of non-competitive NMDA antagonists MK-801
and phencyclidine (PCP) is complemented by NMDA deficits in
post-mortem brain in schizophrenia. In the present study, differential sensitivity to the behavioural effects of MK-801 and PCP was
examined in mice containing partial deletion of the NRG1 gene in
order to elucidate the relationship between NRG1 genotype and the
psychotomimetic effects of NMDA receptor antagonism. Changes in
social behaviour and exploratory activity were assessed in NRG1 mutants vs. wildtypes following acute or subchronic administration of
MK-801 or PCP. Neuregulin-1 mutants and wildtype controls mice
were also examined by magnetic resonance (MR) volumetry using
high-resolution T2-weighted imaging at 7 T in order to examine
effects of NRG1 genotype on presence of schizophrenia-relevant
structural brain endophenotypes. The results indicate that NRG1
knockout mice display (a) altered responsivity to NMDA receptor
antagonists compared to controls (b) volumetric changes in brain
areas implicated in schizophrenia. This work was funded by Science Foundation Ireland & Health Research Board of Ireland.
TVI.31
LOCAL BLOCADE OF NMDA RECEPTORS
IN THE RODENT PREFRONTAL CORTEX MODIFIES
OSCILLATORY ACTIVITY AND c-Fos EXPRESSION
IN THE NUCLEUS ACCUMBENS
Nowak K., Meyza K., Kasicki S., Hunt M.
In humans subanesthetic doses of noncompetitive NMDA receptor
(NMDAR) antagonists can produce symptoms similar to those observed in schizophrenia. In rodents, systemic application of NMDAR antagonists produce hyperactivity, stereotypies and abnormal
social interactions used to model certain features of schizophrenia. It was also shown that NMDAR antagonists induce the high
frequency oscillations (HFO, 140–180 Hz) in the nucleus accumbens (NAc). Several neurophysiological models of schizophrenia
emphasize the role of interactions between frontal cortical areas,
especially medial prefrontal cortex (mPFC) and NAc. The aim of
this study was to examine the effects of local infusion of MK-801
(a selective NMDAR antagonist) in the mPFC on oscillatory activity recorded in the NAc. Experiments were performed on freely
moving rats, bilaterally implanted with guides in the mPFC and
an electrode in the right NAc. Bilateral administration of MK-801
to the mPFC produced a substantial increase in the power of HFO,
which was associated with NMDAR antagonist-induced changes
in locomotor activity and behavior. In another group of animals we
examined the effects of local infusion of MK-801 into mPFC on
c-Fos expression in NAc. Prominent c-Fos response was observed
in the NAc in relation to saline and control groups. These results
suggest that local administration of MK-801 to the mPFC modifies
oscillatory activity and c-Fos expression in the NAc.
TVI.32
CANNABINOID CB1 RECEPTOR ANTAGONIST
RIMONABANT ATTENUATES REINSTATEMENT
OF NICOTINE PLACE PREFERENCE PROVOKED
BY NICOTINE OR MORPHINE IN RATS
Biala G., Budzynska B., Staniak N.
Department of Pharmacology and Pharmacodynamics, Medical
University of Lublin, Lublin, Poland
Reinstatement of drug-seeking behaviour in animals is relevant
to relapse to drug taking in humans. In the present study, we used
the conditioned place preference paradigm to investigate the establishment, extinction, reinstatement and cross-reinstatement of
nicotine-induced place conditioning in rats. We have revealed that
nicotine produced a place preference to the compartment paired
with its injections during conditioning. Once established, nicotine
place preference was extinguished by repeated testing. Following
this extinction phase, nicotine-experienced rats were challenged
with nicotine (0.5 mg/kg) or morphine (10 mg/kg). These priming injections of both drugs renewed a marked preference for
the compartment previously paired with nicotine. In the second
step, we examined the influence of CB1 cannabinoid receptor antagonist rimonabant (0.5, 1 and 2 mg/kg) on the reinstatement of
nicotine place conditioning. It was shown that rimonabant, dosedependently, attenuated the reinstatement of nicotine-conditioned
response. Our findings may further indicate similar endocannabinoid-dependent mechanisms involved in the development of reinstatement of nicotine-conditioned place preference provoked by
priming injections of nicotine and morphine. As reinstatement of
drug-seeking is a factor for the development of drug dependence,
CB1 receptor antagonists may be useful in the relapse-prevention
phase of addiction treatment, including nicotinism and polydrug
abuse.
TVI.33
ADENOSINE RECEPTOR AGONISTS ATTENUATE THE
EXPRESSION OF SENSITIZATION TO MORPHINEINDUCED PLACE PREFERENCE IN RATS
Listos J., Talarek S., Fidecka S.
Behavioural sensitization is an enhancement of certain drug-induced effects which develops following repeated, intermittent exposure to opioids and psychoactive drugs. The sensitization can be
measured in various animal models, e.g. conditioned place prefer-
ence paradigm. In the present studies we undertook to investigate
an involvement of adenosine receptor agonists in expression of
sensitization to morphine-induced rewarding effects in rats. Three,
intraperitoneal (i.p.) injections of morphine (5.0 mg/kg) induced
conditioned place preference. Five days later, in animals with prior
history of morphine administration, ineffective dose of morphine
(0.75 mg/kg i.p.) was administered. That dose was able to induce
a significant intensification of morphine response in conditioned
place preference paradigm. It showed that morphine-induced sensitization to rewarding effects had been developed. To determine
the effects of adenosine receptor agonists on expression of sensitization, selective and non-selective drugs were administered 15 min
before the last injection of morphine. We showed that all adenosine
drugs were able to attenuate the expression of morphine sensitization and the strongest effects were produced by selective (A1) and
non-selective (A1 and A2A) adenosine receptor agonists. We conclude that adenosine agonists may play an important role in drugseeking behaviour underlying the development of addiction.
TVI.34
CHARACTERISTIC OF ALCOHOL-PREFERRING WHP
AND NON-PREFERRING WLP LINES OF RATS
Dyr W.1, Cwiek M.1, Taracha E.2, Kostowski W.1
1
Department of Pharmacology and Physiology of the Nervous
System, 2 Department Neurochemistry, Institute Psychiatry and
Neurology, Warszawa, Poland
The selected lines of rats WHP (Warsaw High Preferring) and WLP
(Warsaw Low Preferring) determine the animal model to investigate
neurobiological mechanism of ethanol action. WHP rats drink excessive amounts of alcohol whereas WLP rats drink only small amounts.
Patterns of ethanol consumption in WHP and WLP lines are constant. Due to cessation of alcohol intake the WHP rats have developed features of withdrawing signs. Level of dopamine and serotonine is lower in some brain regions of the WHP rats. WHP rats show
an increased responsiveness to the stimulatory effects of low dose of
ethanol.It has been suggested that acquisition of acute tolerance to
ethanol may promote increased ethanol consumption. The effects of
acute administration of ethanol (dose – 2.3 g/kg i.p.) were established
by assessing ethanol-induced motor impairment, hypothermia and
blood ethanol level in WHP and WLP rats. The rotarod model was
used to measure motor impairment. Results have been revealed that
WHP rats recovered motor activity and normal temperature when
blood ethanol was at the highest level, indicating the development of
acute tolerance to ethanol. The WLP rats showed similar recovery to
WHP rats but at the lower blood ethanol level.Tolerance to ethanol
may have a great value as a predictor of susceptibility to alcoholism
therefore may significant means in the relation to alcohol consumption and dependence. Generally tolerance is thought as the neuroadaptive processes to intoxicate effect of ethanol.
TVI.35
EFFECT OF NICOTINE ON LOCOMOTION
OF CAENORHABDITIS ELEGANS
Sobkowiak R., Lesicki A.
Faculty of Biology, Department of Cell Biology, Adam Mickiewicz
University, Poznań, Poland
The locomotion of the soil nematode Caenorhabditis elegans is a simple behavior that has been widely used to dissect genetic components
of behavior, synaptic transmission, and muscle function. Numerous
350
studies have reported that exogenous nicotine caused contractions
and spastic paralysis of worms at a concentration of 30 mM. The aim
of this study was to estimate nicotine-induced behavioral response
of C. elegans wild-type (N2) L1 larvae. The L1 larvae were transferred to NGM plates containing 300 μM (–)-nicotine. The Parallel
Worm Tracker was used to measure average locomotion speed and
nicotine-induced paralysis in C. elegans [Ramot et al. (2008) PLoS
ONE 3: e2208). The tracker utilizes a modified web camera (attached
to zoom stereo microscopes), which records the position of tens of
worms in sequential video frames. In each experiment, approximately 40 worms were tracked for 30 s every 10 min. A rapid increase
in the mean speed of worm locomotion (up to 2-fold) was observed
after 10 min of exposure of L1 larvae to nicotine. At the same time,
the lowest number of paralyzed and immobile worms was noticed.
The lowest mean locomotion speed and the maximum of paralyzed
individuals was observed after 40 min of nicotine treatment. It was
followed by a recovery period within 60 min after exposure, during
which the worms acquired tolerance. Further research will be undertaken to analyze the nicotine dose-dependent locomotion response of
all developmental stages of C. elegans.
TVI.36
METABOTROPIC GROUP I GLUTAMATE RECEPTORS
(mGluR) ANTAGONISTS ATTENUATE COCAINEINDUCED TOXICITY AND COCAINE-INDUCED
BEHAVIORAL SENSITIZATION IN MICE
Bochenski M., Kotlinska J.
drugs of abuse. The new analog of deltorphin – DK-4 is a peptide
with high affinity to delta opioid receptors in mouse vas deference
bioassay. The aim of our study was to indicate whether delta opioid
receptors are involved in the rewarding effects of cocaine. Therefore,
we tested influence of (1) opioid receptor antagonists (μ, κ, δ) on the
expression of cocaine-induced conditioned place preference (CPP);
and (2) influence of DK-4 on the cocaine reinstatement of CPP (Sleipness et al. 2007). Rats were conditioned daily with cocaine (5 mg/kg,
i.p.), tested on the 6th day (the expression of CPP), and then retested
for 9 days to monitor the extinction of the CPP. On the day 10 animals
were treated with a single injection of cocaine (5 mg/kg) or DK-4 (5,
10 nmol, i.c.v.) to reinstate the CPP. Our results indicated that naltrindole – a delta opioid receptor antagonist decreased cocaine rewarding
effect (the expression of CPP). DK-4 increased the reinstatement of
cocaine-induced CPP. These studies suggest that delta opioid receptors are involved in the cocaine-induced rewarding effects.
TVI.38
THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM
IN AMPHETAMINE-INDUCED MODULATION OF
NATURAL KILLER CYTOTOXICITY IN RATS
Glac W., Badtke P., Orlikowska A., Tokarski J., Wrona D.
Poland
Cocaine abuse and dependence is a worldwide health problem.
However, there are no currently approved medications to reduce cocaine abuse/relapse and toxicity. Published data showed that group I
mGluR antagonists (mGluR1 and mGluR5) possess an anti-addictive
potential in various animal models of cocaine abuse. In the present
study we assessed the impact of mGluR1 antagonist – EMQMCM
and mGluR5 antagonist – MTEP on the cocaine-induced toxicity (lethality) in mice. Moreover, we evaluated the influence of these antagonists on motivational effect of cocaine measured in the sensitization
test to its hyperlocomotor effect in mice. Our study indicated that
EMQMCM and MTEP, at the doses of 2.5; 5, and 10 mg/kg, dose-dependently decreased cocaine-induced lethality produced by 75 mg/
kg of cocaine. The effect of EMQMCM was stronger than MTEP,
and EMQMCM at the dose of 10 mg/kg completely reduced the lethality induced by cocaine. Furthermore, EMQMCM but not MTEP,
significantly reduced the expression of cocaine-induced (10 mg/kg)
behavioral sensitization. Our results suggest that mGluR1 receptors
are implicated in motivational effect of cocaine but both receptors
(particularly mGluR1) play a role in cocaine-induced toxicity.
It is well known that the main site of the action of amphetamine
(AMPH) are the catecholaminergic neurons located both in central
nervous system and in the sympathetic nervous system (SNS). To
analyse the potential role of the SNS in the mechanism of AMPHinduced changes in natural killer cells cytotoxicity (NKCC) rats
were sympathectomized by 6-hydroxydopamine (6-OHDA, 3 × 50
mg/kg, i.p.) prior to AMPH (1 mg/kg, i.p.) administration. In a separate experiment, rats were pretreated with a α-adrenergic receptor antagonist phentolamine (5 mg/kg, i.p.), β-adrenergic receptor
antagonist propranolol (5 mg/kg, i.p.) or both. NKCC (51Cr-release
assay) and the number of LGL (NK cells) were evaluated in the peripheral blood and spleen. In the peripheral blood AMPH-induced
stimulation of NKCC was completely blocked by 6-OHDA. The
increase in LGL number in the peripheral blood evoked by AMPH
was partially inhibited by sympathectomy. In the spleen both effects of AMPH i.e. reduction of NKCC and decrease in LGL number were completely reversed by 6-OHDA. β-antagonist attenuated the AMPH-induced changes in NKCC and LGL number in
the peripheral blood and spleen. In contrast, changes in NKCC and
LGL number were not affected by α-blockade. These data clearly
show that both AMPH-induced stimulation of the peripheral blood
NKCC and suppression of spleen NKCC are mediated by the SNS.
Furthermore, catecholamines elevated by AMPH modulate the
NKCC via β-adrenergic, but not α-adrenergic mechanism.
TVI.37
ARE DELTA OPIOID RECEPTORS INVOLVED IN THE
REWARDING ACTION OF COCAINE IN RATS?
Pachuta A.1, Gibula-Bruzda E.1, Kunce D.2, Izdebski J.2,
Kotlinska J.1
1
University of Lublin, Lublin, Poland; 2 Department of Chemistry,
Warsaw University, Warszawa, Poland
TVI.39
EFFECTS OF BINGE-LIKE ETHANOL
ADMINISTRATION ON MACROMOLECULE PROFILES
IN HIPPOCAMPUS OF YOUNG ADULT WISTAR RATS
AS ASSESSED BY FT-IR SPECTROSCOPY
Severcan F., Elibol B., Jakubowska Dogru E.
Biological Sciences, Middle East Technical University, Ankara,
Turkey
Delta opioid receptors are involved in modulation of nociception,
thermoregulation, locomotor activity and rewarding properties of
Despite of widespread abuse and the presence of vast data about
ethanol’s adverse effects on behavior, brain morphology and physi-
ology, molecular mechanism underlying deteriorating effects of
chronic ethanol intoxication on the nervous system still remain
elusive. The aim of the present study is revisiting the potential effects of adult alcohol insult and/or withdrawal on the protein, nucleic acids, and lipid profiles in the rat hippocampus by using Fourier
Transform Infrared (FTIR) spectroscopy. It is a rapid, sensitive and
non-disturbing technique which gives information about different
functional groups belonging to biomolecules, simultaneously [Garip
et al. (2007) Appl Spectrosc]. In the current study the rats were intragastrically administered ethanol at a dose increasing from 4.5 g
to12 g/kg for 15 days and then the mean blood alcohol concentration was measured as 605.67 ± 36 mg/dl. FTIR results revealed an
increase in the total content of proteins, lipids, as well as amino,
fatty, and nucleic acids. Moreover, an increase in lipid disordering
and a decrease in membrane fluidity were obtained. Chronic ethanol
intoxication was also found to cause an increase in random coil and
aggregated β sheet content indicating structural changes in proteins,
such as denaturation. Significant variations in DNA and RNA conformation were observed. These molecular changes are discussed in
terms of their potential effects on the neural function.
TVI.40
TETRAHYDROCANNABINOL MAY ENHANCE
NATURAL KILLER CELL CYTOTOXICITY IN RATS
Badtke P., Glac W., Tokarski J.
Poland
It is well known that Δ9- tetrahydrocannabinol (THC), the main active component of marijuana, exerts mainly suppressive influence
on the immune system. Here we present data indicating that THC
induce immune enhancing effects. In this study we examined the
effects of small doses of THC on the natural killer cell cytotoxicity (NKCC) (51Cr – release assay) and also on the number of NK
cells (identified as large granular lymphocytes, LGL) (Timonen
method) in the peripheral blood in rats. Animals received acute
or chronic (10 days) i.p. injections of THC at the doses of 0.2 and
0.5 mg/kg. It was found that acute injection of THC significantly
increased NKCC after the dose of 0.2 mg/kg (+ 131 Δ%; P<0.001
vs. control and + 52 Δ%; P<0.05 vs. 0.5 mg/kg dose). After chronic
exposure to THC, a significant NKCC increase was observed after
the dose of 0.5 mg/kg (+ 209 Δ%; P<0.001 vs. control and + 97
Δ%; P<0.01 vs. 0.2 mg/kg dose). The number of LGL remained
unchanged after both acute and chronic treatment with THC. It
has been reported repeatedly that the doses of over a dozen milligrams of THC suppress NKCC. These results negate the common
opinion about unidirectional suppressive effects of cannabinoids
on the immune system. It seems that the pharmacodynamics rule
of hormesis may also concern the influence of THC on the immune system.
TVI.41
EFFECTS OF CONTINGENT AND NON-CONTINGENT
ADMINISTRATION OF MORPHINE ON THE
DOPAMINE RECEPTORS AND OPIOID PEPTIDES GENE
EXPRESSION IN THE MOUSE BRAIN
Gieryk A.1, Ziolkowska B.1, Touriño C.2, Maldonado R.2,
Przewlocki R.1
1
Pharmacology PAS, Kraków, Poland; 2 Departament of Experimental
and Helath Sciences, Pompeu Fabra University, Barcelona, Spain
Changes in the gene expression produced by drugs of abuse differ
depending on whether the drug is self-administered by the subject (contingent administration) or injected to the animals by the
experimenter (non-contingent administration). To verify whether
expression of the opioid peptide precursors and dopamine receptors depend on the direct pharmacological effect of the drug or
reflect the cognitive processes associated with self-administration
of morphine we employed “yoked” self-administration procedure. The experiment was performed on C57BL/6J mice that were
trained to self-administer morphine. We used the technique of in
situ hybridization to measure the dopamine receptors (D1R and
D2R) and opioid propeptide (proenkephalin and prodynorphin)
mRNA levels in several brain regions implicated in addiction.
Differences in the D1R and D2R gene expression were found in
the dorsal striatum and nucleus accumbens, where an increase of
mRNA levels upon active, but not passive morphine administration was observed. No changes in the expression of both opioid
propeptide genes were detected in all investigated brain regions of
mice receiving contingent or non-contingent morphine injections.
The observed increase in the D1R and D2R genes expression suggests that changes in the dopaminergic system may be specifically
associated with the motivational and cognitive processes underlying self-administration of morphine. Supported by EU grants:
LSHM-CT-2007-037669 and LSHM-CT-2004-005166.
TVI.42
EFFECT OF DAIDZIN AND PUERARIN
ADMINISTRATION ON BEHAVIORAL
AND COGNITIVE FUNCTIONS IN CHRONICALLY
ETHANOL DRINKING OUTBRED RATS
Mikolajczak P.1,2, Okulicz-Kozaryn I.2, Golys K.2,
Moryson E.2, Kaminska E.2, Szulc M.2,
Bobkiewicz-Kozlowska T.2, Mrozikiewicz P.1
1
Department of Pharmacology and Biotechnology Branch of
Medicinal Plants, Institute of Natural Fibres and Medicinal Plants,
Poznań, Poland; 2 Department of Pharmacology, Poznan University
of Medical Sciences, Poznań, Poland
Administration of isoflavones present in Pueraria radix, daidzin
(DAI) or puerarin (PUE), reduces ethanol intake in rodents. The paradigm showed by these compounds is probably mediated centrally,
therefore an assessment of influence of DAI and PUE on association
between alcohol consumption and changes in memory and motor
functions seems to be of scientific importance. Ethanol “preferring”
(PR) and “non-preferring” (NP) rats were treated with DAI (40 mg/
kg, p.o.) and PUE (150 mg/kg, p.o.) for 28 consecutive days and their
motor activity, motor coordination, anxiety-related reactions, shortand long-term memory were assessed. It was found that DAI (23%)
and PUE (52%) lowered alcohol intake in PR rats. PUE produced a
facilitation effect on long- (significantly) and short-term (tendency)
memory in PR rats. The effects seemed to be specific since PUE
did not affect motor activity and anxiety-related reactions and led to
improvement motor coordination in PR rats. On the contrary, DAI
did not change cognitive and behavioral functions in PR animals,
whereas positive effect especially on short-term memory in NP rats
was found. Unfortunately, DAI lowered motor activity and coordination in NP animals. In conclusion, since PUE attenuated the deficits of inhibitory avoidance performance induced dizocilpine (Hsieh
et al. 2002), therefore its positive effect on long-term memory via
NMDA receptor activation in PR rats can be considered. The study
has been supported by grant N405024 32/1684.
352
TVI.43
EFFECTS OF CANNABIDIOL ADMINISTRATION
ON BODY WEIGHT GAINS IN RATS
Ignatowska-Jankowska B., Jankowski M., Swiergiel A.
Poland
Cannabidiol (CBD), the major non-psychoactive compound of
Cannabis has been reported to display neuroprotective, anti-inflammatory, and anti-tumor actions. Despite growing interest in
pharmacological effects of CBD, there is no data concerned with
the effects of CBD on body weight, food intake or metabolism.
The present study examined whether CBD administration affects
body weight, during 13 days of systemic CBD treatment. Adult
male Wistar rats (n=54), weighing approximately 250 ± 20 g at the
beginning of the experiment received intraperitoneal injections of
CBD at dose of 2.5 or 5 mg/kg/day, or the vehicle, for 13 consecutive days. The animals were weighed each day 2 h before each
injection. Body weight on the first day of treatment served as baseline (100%), then in following 13 days percentage body weight
gains were calculated. Repeated CBD administration significantly
slowed body weight gains. Two-way ANOVA revealed significant
difference between weight gains of rats receiving CBD and the
control group (P<0.0001).Rats receiving CBD at dose of 5 mg/kg
gained, on average, 8.0 ± 0.9 g (SEM) less weight than control
animals, which is 19.6% of total weight gain of controls during
that period. Mean body weight gain of rats receiving 2.5 mg/kg
of CBD was 6.8 ± 0.9 g lower than controls (16.6% of total weight
gain of controls).The results indicate that CBD administered in
low doses lowers body weight gain. The data suggest that CBD
may affect food intake or metabolism.
TVI.44
WARSAW HIGH ALCOHOL-PREFERRING (WHP) AND
LOW ALCOHOL-PREFERRING (WLP) RATS DIFFER IN
BEHAVIORAL AND MIDBRAIN DOPAMINE SYSTEMS’
RESPONSES TO MORPHINE
Plaznik A.1, Taracha E.1, Dyr W.2, Cwiek M.2, Chrapusta S.3,
Turzynska D.1, Sobolewska A.1, Bidzinski A.1, Walkowiak J.1
1
Department of Neurochemistry, 2 Department of Pharmacology
and Physiology of the Nervous System, Institute of Psychiatry
and Neurology, Warszawa, Poland; 3 Department of Experimental
Pharmacology, Mossakowski Medical Research Centre PAS,
Warszawa, Poland
Rats were given 14 “daily” (6 doses/week) s.c. doses of 0.9% NaCl (Sal),
morphine (Mor), or methadone (Met), then left untreated for 14 days, and
finally challenged with Mor, except that half each of the Sal groups were
given Sal instead. All the rats were then tested for open field behavior, and
were sacrificed 2 h post-challenge. Striatal (CPu), accumbal (Acb), sensorimotor cortex (S1) and prefrontal cortex contents of dopamine (DA),
3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA)
and 3-methoxytyramine (3 MT, assessed only in the CPu and Acb) were
measured by HPLC. Analysis of total distance covered by the rats showed
Mor and Met-induced sensitization to Mor in the WHP rats only, whereas
the distance covered in the central part of the test arena was significantly
affected (increased) only by the Mor pretreatment and only in the WHP
rats. There was no significant between-line or treatment-related difference
in DA content, and no significant between-line difference in baseline DA
metabolite contents, except that S1 HVA content was significantly higher
in drug-naive WLP rats than in drug-naive WHP rats. HVA and – to a
lesser extent – DOPAC contents were, in general, higher in the Mor-challenged rats than in the respective Sal-treated controls, but the differences
were more pronounced in the WLP rats than in their WHP counterparts,
whereas the opioid -pretreated WHP rats showed higher CPu and Acb 3
MT contents than their WLP counterparts.
TVI.45
CHANGES OF ERK1 AND ERK2 PHOSPHORYLATION
IN HIPPOCAMPUS DURING MORPHINE-INDUCED
CONDITIONED PLACE PREFERENCE IN MOUSE
BRAIN
Wawrzczak-Bargiela A., Bilecki W., Przewlocki R.
Pharmacology, Kraków, Poland
Extracellular signal-regulated kinases (ERK1 and ERK2) may
play an important role in the molecular mechanisms of opiates addiction. Recently, the role of hippocampus in the process of addiction has attracted an attention as addictive drugs like morphine
may affect the normal function of this region leading to a formation of aberrant learning. Our previous results demonstrated that
acute and prolonged morphine treatment leads to changes of ERK1
and ERK2 phosphorylation. However, the specific pattern of alterations of ERK1 and ERK2 phosphorylation (which is required
for ERKs activity) in the hippocampus, during the development
of morphine reward has not been studied yet. We discovered that
first morphine conditioning increased ERK1 and ERK2 phosphorylation in CPP paradigm, but after third morphine session during
CPP, we observed the decrease of ERK1 and ERK2 phosphorylation. Moreover, an increase of pERK2/pERK1 ratio correlated linearly with the expression of place preference following morphine
administration. The changes of phosphorylation were observed in
mossy fibers, the structure, which is involved in spatial learning
and memory. In hippocampus, the observed changes of ERK phosphorylation favoring mainly ERK2 activity together with the putative role of ERK2 in learning and long-term memory suggest that
morphine-induced interactions within ERK pathway participate
in reward-controlled learning. Research supported by Ministry of
Science and Education Grant N401 066 31/168.
TVI.46
INTRAVENOUS COCAINE ALTERS LYMPHOCYTE
SUBSETS DISTRIBUTION IN PERIPHERAL BLOOD
OF RATS
Jankowski M., Ignatowska-Jankowska B., Glac W., Swiergiel A.
Poland
Cocaine use has been associated with the increased risk for HIV
and HCV infection in humans. The addicts have faster progression
of HIV infection and increased incidence of AIDS suggesting that
cocaine use results in a specific functional impairment of the immune system. Present study aimed to evaluate lymphocyte subsets
number and distribution in peripheral blood after acute series of
intravenous cocaine infusions. Adult male Wistar rats received 3
injections of cocaine hydrochloride (5 mg/kg/ml) in 30 min intervals. Blood samples were collected 30 min after the last infusion. Total leukocyte number, percentage numbers of leukocyte
subpopulations and T, B, NK, T helper and T cytotoxic lymphocyte subsets were assessed. Intravenous cocaine caused significant decrease in total leukocyte and lymphocyte number and sig-
nificant fall in total numbers of T, B, NK and both T helper and T
cytotoxic lymphocyte subsets. Furthermore, intravenous cocaine
altered proportions between lymphocyte subsets by increasing T,
mainly T helper and decreasing B, T cytotoxic and NK lymphocyte percentage portion. Immunosuppressive effect of intravenous
cocaine infusions may thus be connected with alterations in lymphocyte subsets proportions. Such destabilization of lymphocyte
subsets balance, especially T helper lymphocyte, could constitute
basis of increased susceptibility for HIV and HCV infections.
TVI.47
GENE PROFILING THE REWARDING EFFECT
OF MORPHINE IN STRIATUM AND HIPPOCAMPUS
Bilecki W., Przewlocki R.
Persistent changes that take place during the development of opioid addiction are thought to be due to adaptive changes in gene
expression in the brain. In the present study we used DNA microarrays (Illumina) to analyze the expression of about 45 000
transcripts in the selected brain regions (striatum and hippocampus) of C57Bl/6J mice during the morphine-induced conditioned
place preference (CPP) test. Repetitive morphine treatment during
morphine conditioning led to an altered expression of 257 genes.
Many of these genes coded for heat shock proteins, neuronal transport and kinases including mitogen-activated protein kinases. The
observed alterations in the gene transcription resulted from the
morphine-induced conditioning rather than CPP test alone. The
genes meeting criteria for statistical significance revealed strong
correlation coefficients with the expression of place preference
(measured as increased time spent on the morphine-paired side).
These findings suggest that morphine-conditioning induces the
persistent changes in gene transcription that correlate with the rewarding behavior. Research supported by Ministry of Science and
Education Grant N401 066 31/168.
TVI.48
EVALUATION OF SPONTANEOUS PARTIAL
WİTHDRAWAL AND EFFECT OF CLONIDINE
IN INTERMITTENT MORPHINE ADMINISTRATION
IN RATS
Enginar N., Hatipoğlu I., Allahverdiyev O.
Istanbul Faculty of Medicine, Department of Pharmacology
and Clinical Pharmacology, Istanbul University, Istanbul, Turkey
Using novelty-induced grooming as a marker of stress response
and clonidine as an anti-stress drug, present study tested the hypothesis that intermittent injection of morphine may induce stress
and allow some withdrawal during each interval (Houshyar et al.
2003). Male Wistar rats were injected s.c. with saline or morphine
twice daily (08:30 and 18:30) for 4 days with increasing doses (10,
20, 30, 40 mg/kg/injection/day). On days 1–4, animals were given
tap water with or without 5 μg/ml clonidine (18:30–8:30). On day
5, at the time of morning injections, animals were observed for
5 minutes for grooming and spontaneous withdrawal [14-h withdrawal assessed with global withdrawal score (GWS)]. Then, animals were injected s.c. with morphine (40 mg/kg) or saline and 2
hours later with naloxone (1 mg/kg) and observed for 15 minutes
for withdrawal symptoms. Data were analyzed using ANOVA and
Bonferroni test. Morphine and morphine+clonidine groups spent
less time in grooming and produced marked GWS and naloxoneprecipitated withdrawal signs. Present study could not demonstrate an increase in grooming reflecting stress before the next
dose of intermittent use of morphine, but provides evidence that
the injection regimen allows manifestations of partial morphine
withdrawal. Clonidine, possibly due to insufficient water intake,
could not prevent withdrawal signs. Further experimentation may
provide more data to clarify spontaneous withdrawal associated
with the treatment regimen.
TVI.49
EXTRACELLULAR CORTICAL SEROTONIN AND
DEPRESSION-RELATED BEHAVIOUR IN THE FORCED
SWIM TEST IS INFLUENCED BY INTERLEUKIN-2
Karrenbauer B.1, Mueller C.2, Schwarting R.1, Spanagel R.3,
Pawlak C.3
1
Department of Physiological Psychology, Philipps University
Marburg, Marburg, Germany; 2 Institute of Psychiatry, King’s
College, London, UK; 3 Department of Psychopharmacology,
Central Institute of Mental Health, Mannheim, Germany
It is assumed that cytokines (like interleukins) can influence depression and anxiety. Interleukin-2 (IL-2) is suggested
to be one factor, which may mediate behavioural and neurochemical (e.g., serotonergic) features of depression in the brain.
Previously, we have shown in rats that IL-2 mRNA in the striatum
and prefrontal cortex is correlated with anxiety-like avoidance behaviour in an elevated plus-maze. Additionally, striatal IL-2 microinjections affected anxiety-like behaviour in a biphasic way. In
the present study, we investigated the impact of systemically (i.p.)
injected IL-2 (2.5 μg/kg) on serotonegic (5-HT) and dopaminergic
neurotransmission in various cortical areas by in-vivo microdialysis
in anaesthetised rats (Exp. 1). Furthermore, based on the serotonergic time profile, we conducted two experiments to test for delayed
(test 2 h post injection, Exp. 2) and acute (Exp. 3) behavioural effects of systemic IL-2 (0–5 μg/kg) on depression-related behaviour
in a forced swim test (FST). The neurochemical results revealed
that systemic IL-2 lastingly reduced extracellular 5-HT levels in
the medial prefrontal (−75%), occipital (−70%), and temporal cortices (−45%). In contrast, dopamine was moderately reduced only
in the medial prefrontal cortex. The functional relevance of these
specific neurochemical changes were supported by the subsequent
behavioural evaluation showing IL-2 dose-dependent effects on
depression-related behaviour in the FST after delayed testing.
TVI.50
THE INVOLVEMENT OF NITRIC OXIDE (NO)
IN THE AMNESIC EFFECTS OF DIAZEPAM IN MICE
Talarek S., Orzelska J., Listos J., Fidecka S.
Chair and Department of Pharmacology and Pharmacodynamics,
Medical University of Lublin, Lublin, Poland
Literature data show the relationship between L-arginine:NO:cGMP
pathway and g-aminobutyric acid (GABA)-mediated transmission in the central nervous system. Benzodiazepines are known
to enhance the GABA-ergic neurotransmission and well established as inhibitory modulators of memory processing. More-
354
over, the role of NO in learning and memory processes has been
proposed. The present studies were designed to evaluate the role
of L-arginine:NO:cGMP pathway in the amnesic effects of diazepam (DZ) in the modified elevated plus-maze (mEPM) task in
mice. Our experiments indicated that DZ (1 mg/kg, s.c.) impaired
elevated plus-maze memory performance in mice. Pretreatment
with L-arginine, the NO precursor, (500 mg/kg, i.p.) prevented the
amnesic properties of DZ. While, 7-nitroindazole (7-NI), the neuronal NO synthase inhibitor (nNOS), (40 mg/kg, i.p.) and methylene blue (MB), the soluble guanylate cyclase (sGC) inhibitor,
(5 mg/kg, i.p.) enhanced the DZ-induced memory deficits. Moreover, the effect of both 7-NI and MB were reversed by L-arginine
(250 mg/kg, i.p.). It is important to note that presented data are not
due to either 7-NI-, MB-induced impairment of memory or changes in locomotor activity, because 7-NI and MB given alone, had no
impact on the mEPM behaviour and locomotor activity of mice.
Taken together, these results suggest that an inhibition of the Larginine:NO:cGMP pathway may be involved in the amnesic effects of benzodiazepines.
TVI.51
INTERSTRAIN DIFFERENCES IN 5-HT3 RECEPTOR
CENTRAL-INDUCED HYPOTHERMIA AND
COMPARISON WITH HYPOTHERMIA MEDIATED
VIA 5-HT1A RECEPTOR
Kondaurova E., Naumenko V.
Laboratory of Behavioral Neurogenomics, Institute of Cytology and
Genetics SB RAS, Novosibirsk, Russia
The selective agonist of 5-HT3 receptors 1-(3-Chlorophenyl)biguanide hydrochloride (m-CPBG) administered intraventricularly
(40, 80 or 160 nmol) produced distinct hypothermic response in
AKR/2J mice. At the same time, intraperitoneal administration of
m-CPBG in wide dose ranges (0.5, 1.0, 5.0 or 10.0 mg/kg) caused
no effect on the body temperature. These findings suggest: (1)
the implication of central, rather than peripheral 5-HT3 receptor in thermoregulation; (2) the inability of m-CPBG to permeate through blood-brain barrier in mice. The comparison of brain
5-HT3-induced hypothermic reaction between six inbred mouse
strains (DBA/2J, CBA/Lac, C57BL/6, BALB/c, ICR, AKR/J) was
performed and two highly sensitive to m-CPBG mouse strains
(CBA/Lac and C57BL/6) were found.In aforenamed six inbred
mouse strains the functional activity of 5-HT1A receptors was
studied. Considerable interstrain differences were found in the
intensity of hypothermic response produced by intraperitoneal
administration of selective agonist of 5-HT1A receptors – 8-OHDPAT. The comparison of hypothermic reaction induced by central administration of m-CPBG and hypothermia produced by
8-OH-DPAT revealed significant correlation between 5-HT3 and
5-HT1A-induced hypothermia in five out of six investigated inbred
mouse strains. The data suggest the association of the brain 5-HT3
and 5-HT1A receptors in the mechanisms of serotonin-conditioned
hypothermia.
TVI.52
EVIDENCE FOR REGIONALLY SPECIFIC CHANGES
IN HIGH-FREQUENCY OSCILLATIONS AFTER
INJECTION OF KETAMINE IN FREELY MOVING RATS
Hunt M., Falinska M., Leski S., Wojcik D., Kasicki S.
Department of Neurophysiology, Nencki Institute of Experimental Biology PAS, Warszawa, Poland
We have shown previously that the NMDAR antagonists ketamine and MK-801 enhance high-frequency oscillations (140–
180 Hz, HFO) in the rat nucleus accumbens (NAc). However, it
is not known whether NMDAR antagonists can modify HFO
recorded in structures outside the NAc. Thus we have examined
the effect of a single subanesthetic dose of ketamine (25 mg/kg)
on oscillatory activity in local field potentials recorded in the
neuroanatomically related dorsal striatum and in the hippocampus, where spontaneous high-frequency oscillations (ripples)
have been well described. We used both monopolar and bipolar recordings to evaluate oscillatory activity recorded at baseline and after injection of ketamine. In monopolar recordings
ketamine-induced increases in the power of HFO were present
in all structures, although the power was always substantially
larger in the NAc. Bipolar recordings, known to remove common-mode input, were used in an attempt to more precisely localise the source of HFO. In all cases ketamine-induced HFO
were still present in the signals recorded from the NAc, but not
from the dorsal striatum or hippocampus. Notably, spontaneous
sharp-wave ripples also remained in the bipolar signal from the
hippocampus. In a separate study of the depth-profile analysis
of oscillatory activity we found the power of HFO was substantially larger in areas closest to the NAc. These fi ndings suggest
that ketamine may produce some regionally specific changes in
HFO.
TVI.53
STATE-DEPENDENT MODIFICATION OF HIGHFREQUENCY OSCILLATIONS IN THE RODENT
NUCLEUS ACCUMBENS
Hunt M.1, Matulewicz P.1, Gottesmann C.2, Kasicki S.1
1
Biology PAS, Warszawa, Poland; 2 Department of Biology, Faculty od
Science, University of Nice-Sophia Antipolis, Nice, France
Spontaneous high frequency oscillations (HFO) in the local
field potential recorded in the nucleus accumbens (NAc) are
typically represented by a small peak in the power spectra in
the range of 140–180 Hz. These HFO are known to occur in the
awake state, but their distribution over the sleep-wake cycle has
not been investigated. To address this issue we fi rstly examined
the power of HFO during periods of quiet waking, slow-wave
sleep (SWS) and REM sleep. Since the electrophysiological
activity during general anesthesia resembles certain features
of naturally occurring SWS we went on to examine the effect
of pentobarbital, isoflurane or urethane anesthesia on spontaneous and ketamine-induced increases in HFO. We found that
the power of spontaneous HFO decreased significantly during
periods of SWS with respect to both quiet waking and REM
sleep. General anesthetics also reduced the power of spontaneous HFO recorded in the NAc and completely blocked the
ketamine-induced increase. These fi ndings suggest that behavioural states where the generation of mental activity is most
intense are associated with the presence of HFO in the NAc. In
line with this, states which lead to decreased mentation, such
as naturally occurring SWS and general anesthesia are associated with reductions in the power of HFO. Together these fi ndings suggest that the presence of HFO may have behaviourally
meaningful consequences.
TVI.54
EFFECT OF CO-TREATMENT WITH FLUOXETINE
AND AMANTADINE ON THE BEHAVIORAL AND
IMMUNOLOGICAL PARAMETERS OF RATS
SUBJECTED TO THE FORCED SWIMMING TEST
Rogoz Z.1, Kubera M.2, Basta-Kaim A.2
1
Department of Pharmacology, Institute of Pharmacology
PAS, Kraków, Poland 2 Department of Experimental
Neuroendocrinology, Institute of Pharmacology PAS, Kraków,
Poland
was even stronger in TCRd knockout mice. On the other hand in
b2m-/- and CD1d-/- mice the prolonged antidepressant administration did not affect CS reaction. PCL sensitization significantly increased spleen weight and proliferative activity of splenocytes in
B10PL, TCRd-/- and b2m-/- mice. Prolonged antidepressant treatment attenuated this effect but only in B10PL mice. Presented data
might suggest that inhibitory effect of desipramine and fluoxetine
on CS reaction could be caused by their influence on CD8+ and
NKT regulatory cells that attenuate T cell-mediated immune response.
Considerable attention has been paid to a possible role of immunological dysregulation in the pathogenesis of depression. It has been
reported that combined administration of antidepressant drugs
and the non-competitive NMDA receptor antagonist amantadine
(AMA) reduces immobility time in the forced swimming test.
Moreover, preliminary clinical data show that such a combination
of drugs has beneficial effects in treatment-resistant depressed patients. Since immune activation and a pro-inflammatory response
are clearly evident in treatment-resistant depression, the aim of
the present study was to examine the effect of an antidepressant
combination of FLU and AMA on immune parameters in rats subjected to the forced swimming test. The obtained results revealed
synergistic antidepressant effects of combined administration of
FLU (10 mg/kg) and AMA (10 mg/kg) – drugs otherwise ineffective when given separately in such doses. The antidepressant
activity was accompanied with a reversal of the stress-induced
increase in the proliferation of splenocytes in response to concanavalin A, and a significant enhancement in the production of the
negative immunoregulator interleukine-10. Moreover, the relative
spleen weight in these rats was also reduced after joint administration of FLU with AMA in comparison with rats treated with the
vehicle. In summary, the antidepressive efficacy of a combination
of FLU and AMA given in suboptimal doses may be related to
their negative immune effects.
TVI.56
EFFECT OF BACTERIAL LIPOPOLYSACCHARIDE
AND INTERLEUKINE 6 ON CATALEPSY AND
SPONTANEOUS LOCOMOTION IN MICE
Bazovkina D., Kulikov A.
Laboratory of Behavioral Neurogenomics, Institute of Cytology
and Genetics, SB RAS, Novosibirsk, Russia
TVI.55
THE EFFECT OF ANTIDEPRESSANT DRUGS ON
DELAYED-TYPE HYPERSENSITIVITY REACTION
IN BETA2M-/-, TCRDELTA-/- AND CD1D-/- MICE
Kubera M.1, Szczepanik M.2, Majewska M.2, Zemelka M.2,
Leskiewicz M.1, Basta-Kaim A.1, Budziszewska B.1, Grygier B.1,
Regulska M.1, Korzeniak B.1, Jagla G.3, Nowak W.3, Lason W.1
1
Pharmacology PAS, Kraków, Poland; 2 Department of Human
Developmental Biology, 3 Department of Pain Treatment and
Palliative Care, Jagiellonian University Medical College, Kraków,
Poland
Delayed-type hypersensitivity (DTH) is a T cell-mediated immune reaction that plays a major role in the pathogenesis of various inflammatory disorders. One of the most characteristic DTH
phenomena is contact hypersensitivity (CS) used to assess cellmediated immunity against tumor cells and microbes that survive
within macrophages. Recently it was suggested that activation of
immune system plays a role in etiology of depression and that antidepressive agents have negative immunoregulatory effects. Our
present studies showed that chronic fluoxetine and desipramine
administration significantly inhibited CS reaction to picryl chloride (PCL) in B10.PLwild type mice (by 58% and 48%, respectively, when compared to positive control) and their inhibitory effect
Catalepsy is a state of pronounced motor inhibition and is found in
all vertebrates. The exaggerated form of catalepsy is a syndrome
of some grave mental disorders in human. The selective breeding
for high predisposition to catalepsy showed that the major gene
of catalepsy is located on the distal fragment of chromosome 13
and seems to be associatied with the Il6st gene coding the gp130
protein, which is the shared protein in the signal transduction
from interleukins, participating in the cell differentiation, immune
and endocrine regulation. Experiments were carried out on adult
C57BL/6J catalepsy-resistant mouse males. Bacterial lipopolysaccharide (LPS; 100, 200 μg/kg, i.p.) significantly depressed all activity in the open-field and it induced cataleptic immobility (more
than 20 s three times) in 50% of mice in each group. Interleukine
6 (IL-6; 200ng/mouse, i.p.) did not effect on behavior in openfield, but it induced immobility in 70% of mice. Administration
of LPS or IL-6 increases the level of IL-6 which binds to the IL-6
receptor, this reaction results to activation of gp130 protein. So,
caleptogenic effect of LPS and IL-6 is in good correlation with
association of predisposition to catalepsy with Il6st gene. Since
depressive-like behavior and immunity disturbances were found
in mice selected for catalepsy and LPS treatment could model depression, gp130 protein seems to be involved in regulation of both
depression and catalepsy.
TVI.57
STUDY ON THE POTENTIAL EFFECTS OF FETAL
EXPOSURE TO DICLOFENAC SODIUM ON BRAIN
MORPHOLOGY AND BEHAVIOR IN YOUNG WISTAR
RATS
Jakubowska-Doğru E.1, Elibol B.1, Arıkan B.2, Sezer M.1,
Kaya Z.1, Tufanlı Ö.1, Ragbetli M.3, Kaplan S.2
1
Department of Biological Sciences, Middle-East Tchnical University,
Ankara, Turkey; 2 Department of Histology and Embryology,
Ondokuz Mayis University, School of Medicine, Samsun,
Turkey; 3 Department of Histology and Embryology, Yuzuncu Yıl
University, School of Medicine, Van, Turkey
Diclofenac sodium (DS) acts as a potent cyclooxygenase inhibititor, reducing arachidonic acid release and prostaglandins formation. It is commonly used as a non-steroid anti-inflammatory drug
and the potential adverse effects of its administration during pregnancy are of medical and public concern. In the present study, the
356
DS in a dose of 1 mg/kg/day was subcutaneously injected to the
pregnant Wistar rats throughout 5–20 gestational days. One group
of 4-week old pups was sacrificed and the counts of principal neurons in cerebellum and hippocampus were done using stereological methods. Another group of pups was subjected to a battery
of behavioral tasks. A significant decrease in the total number of
Purkinje cells but not CA pyramidal neurons was observed in the
drug-treated juvenile pups. No substantial between-group differences were found in most of the applied behavioral tasks including
plus maze anxiety test, and learning/memory tasks such as 12-arm
radial maze and the Morris water maze, the latter run both under
allo- and idiothetic stimulus conditions. Only in the Open Field
test, fetal DS male but not female pups showed slower rate of habituation compared to controls. The obtained results indicated that
the moderate doses of DS administered between 5–20 gestation
days in rats equivalent of the second half of the 1st and the whole
2nd trimester in human) resulted in some neuronal losses which,
however, had no adverse behavioral impact.
TVI.58
(+)-UH 232, A PARTIAL AGONIST OF THE D3
DOPAMINE RECEPTORS, ATTENUATES COGNITIVE
EFFECTS OF ANGIOTENSIN IV AND
des-Phe6 -ANGIOTENSIN IV IN RATS
Braszko J.
Department of Clinical Farmacology, Medical University
of Bialystok, Białystok, Poland
We have recently found that the postsynaptic D3 dopamine (DA) receptors appear not to participate in the memory enhancing effects of
the angiotensin AT4 receptor agonists angiotensin IV (Ang IV) and
des-Phe6-Ang IV. In this study we evaluated role of the presynaptic
DA D3 receptors in these effects. For that purpose effects of (+)-UH
232, a selective D3 DA receptors partial agonist preferring presynaptic sites, on the pro-cognitive action of intracerebroventricularly (icv)
injected Ang IV and des-Phe6-Ang IV were examined. Male Wistar
rats weighing 180–200 g were used. Both peptides given at the dose
of 1 nmol facilitated recall of a passive avoidance (PA) behaviour,
improved object recognition (OR), and increased apomorphineinduced stereotype behaviour. In the auxiliary tests performed to
control for the unspecific influence of motor (open field, OF) and
emotional (“plus” maze, PM) effects of our treatments on the results
of memory tests they had either no (OF) or negligible (PM) effects.
Intraperitoneal pre-treatment of the animals with an ineffective on
its own dose (1 mg/kg) of (+)-UH 232 abolished or markedly diminished effects of both peptides on PA and OR but did not influence
enhancement of stereotypy caused by the peptides. These results indicate that the functional presynaptic DA D3 receptors are necessary
for the pro-cognitive effects of Ang IV to occur.
TVI.59
THE GENETIC BACKGROUND IS RESPONSIBLE
FOR RESPONSE TO NATURAL REWARD
Kubik J., Solecki W., Przewlocki R.
The increased inclination to natural rewards like food and sensation seeking have been associated with elevated drug intake in
humans. We aimed to compare four inbred mouse strains with
known different susceptibility to drug addiction in respect to
response to natural rewards. In this study, we investigated four
inbred strains C57Bl/6J, DBA/2J, SWR/J and 129P3/J in the selfadministration of food (SAF) and operant sensation seeking (OSS)
models in three paradigms: (1) the fixed ratio operant task (FR),
(2) the progressive ratio operant task and (3) the concurrent choice
operant task. No difference in response to reward in SAF FR paradigm was found among the studied strains. In contrast C57Bl/6J
mice performed better then DBA/2J and 129P3/J strains in OSS
paradigm while SWR mice did not react at all. In turn, the highest score of correct choice in SAF was observed in 129P3/J mice.
The systemic administration of dopamine D1 receptor antagonist
SCH 23390 or opioid receptor antagonists (naloxone or naltrexone) reduced the response to reward in the C57Bl/6J and DBA/2J
mice. Our data suggest that genotype background is responsible
for reward-seeking in SAF and OSS models with C57Bl/6J mice
being the most responsive to the reward. We observed also that
food as a reward has different motivational value than sensation
seeking. Also our study indicates that opioid and dopaminergic
systems may be involved in the response to natural rewards.
TVII: Homeostatic and Neuroendocrine Systems
TVII.01
BRAIN RESPONSE TO PERIPHERAL TUMORS IN RATS
– THE STUDY OF NEUROBIOLOGY OF CANCER
Mravec B.1, Lackovicova L.2, Bundzikova J.2, Bizik J.3, Hulin I.1,
Kiss A.2
1
Institute of Pathophysiology, Faculty of Medicine, Comenius
University, Bratislava, Slovak Republic; 2 Laboratory of Functional
Neuromorphology, Institute of Experimental Endocrinology SAS,
3
Laboratory of Tumor Cell Biology, Cancer Research Institute SAS,
Bratislava, Slovak Republic
A significant amount of data has emerged supporting the hypothesis that the central nervous system might monitor and modulate
tumor growth. The aim of our study was to investigate whether
intraperitoneal tumor growth may induce detectable changes in
brain structures that are involved in the response to immune challenges. Using Fos immunohistochemistry we investigated the
effect of a tumor induced by a single intraperitoneal injection of
BP6-TU2 fibrosarcoma cells to male Wistar rats on the activity
of selected brain structures. Twenty eight days after the tumor
cells administration we found an increased Fos expression in the
nucleus tractus solitarii/A2, A1 noradrenergic cells, parabrachial
nucleus as well as in the hypothalamic paraventricular, supraoptic
and accessory neurons. These structures are involved in the processing of signals related to immune challenges and consequent
elaboration of neuro-endocrine responses. Obtained data supports
the view that the signals related to peripheral tumor development
might be transmitted to the brain. Further studies are necessary to
reveal whether our findings can be attributed to a specific effect
of cancer or whether observed changes in the activity of neuronal
structures reflex processes that only accompany the cancer progression.
TVII.02
HYPOTHALAMIC CRH AND VASOPRESSIN
EXPRESSION IN ANIMAL MODELS OF DEPRESSION
AND ANXIETY
Mironova V., Rybnikova E.
Neuroendocrinology, Pavlov Institute of Physiology RAS,
Saint-Petersburg, Russia
Hypersecretion of the hypothalamic corticotropin-releasing hormone (CRH), the main neuroendocrine hypothalamic-pituitaryadrenal (HPA) axis stimulator, is supposed to be the reason of HPA
axis abnormalities observed in depression and anxiety. It was also
suggested that vasopressin as the major CRH secretagogue might
be involved in HPA axis hyperactivity described in depression. But
neuroendocrine mechanisms of depression and anxiety are still not
revealed. The aim of the present study was to elucidate the role of
hypothalamic CRH- and vasopressin-producing centres in the development of depressive- and anxiety-like states in rats. Animal
models have been applied, including the “learned helplessness” as a
model of depression and Time-dependent sensitization paradigm as
a model of anxiety disorder (posttraumatic stress disorder, PTSD, in
particular). The depressive- and anxiety-like states in rats are characterized by significant increase of CRH-immunoreactivity in the
parvocellular division of the paraventricular nucleus (PVN). Overexpression of CRH in the magnocellular PVN is involved in the
development of anxiety- but not depressive-like state. Anxiety-like
state in rats is accompanied by increase of vasopressin expression
in the magnocellular part of the PVN, suggesting its implication in
the pathogenesis of PTSD. Our data give evidence of common and
specific mechanisms of depression and anxiety and might be useful
in working out new approaches in treatment of these disorders.
TVII.03
STRESS-INDUCED CHANGES IN GENE
EXPRESSION OF CATECHOLAMINE ENZYMES AND
ADRENOCEPTORS IN HEART OF SHAM-OPERATED
AND PVN-DEAFFERENTATED RATS
Bohacova V.1, Mravec B.2, Laukova M.1, Kvetnansky R.1
1
Laboratory of Stress Research, 2 Laboratory of Functional
Morphology, Institute of Experimental Endocrinology SAS,
Bratislava, Slovak Republic
The paraventricular nucleus of the hypothalamus (PVN) regulates
neuroendocrine, autonomic and cardiovascular responses to stress.
Stressors activate catacholaminergic systems which consequently
modulates activity of organism via adrenergic receptors (ARs). Our
work was focused on investigation of changes in gene expression of
epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY) and adrenoceptors in
the heart of sham-operated and PVN-deafferentated rats exposed
to immobilization stress (IMO). PNMT and NPY mRNA levels in
the heart of sham-operated rats were not significantly increased
after a single IMO. However, posterolateral deafferentation of the
PVN reduced PNMT mRNA, whereas levels of NPY mRNA were
elevated. These data suggest a stimulatory role of PVN on PNMT
and an inhibitory role on NPY gene expression. Moreover, β1-AR
mRNA levels were significantly reduced in heart of PVN-deafferentated animals comparing to sham-operated rats. On other side,
after single IMO β1-AR mRNA levels were significantly increased
in PVN-deafferentated rats. Gene expression of other ARs (β2,
β3, α1B) in hearts of stressed PVN-deafferentated animals were
decreased. The found down-regulation of gene expression of ARs
might suggest a rise in sympathetic activity induced by PVN deafferentation. Our data suggest that PVN plays an important role in
stress-induced activation of cardiac sympathetic system.
TVII.04
PROPHYLACTIC USE OF DESIPRAMINE IN WISTAR
RATS WITH HIGH STRESS-SENSITIVITY INCREASES
RESISTANCE TO EXPERIMENTAL TUMOR
METASTASIS
Kubera M.1, Grygier B.1, Lewandowska D.2, Gruca P.3, Rogoz Z.3,
Budziszewska B.1, Basta-Kaim A.1, Leskiewicz M.1, Korzeniak B.1,
Jagla G.4, Nowak W.4, Lason W.1
1
Pharmacology PAS, Kraków, Poland; 2 Department of Animal
Physiology, Gdansk University, Gdańsk, Poland; 3 Department of
Pharmacology, Institute of Pharmacology PAS, Kraków, Poland;
4
Department of Pain Treatment and Palliative Care, Jagiellonian
University Medical College, Kraków, Poland
The effect of antidepressant drugs on tumour progress in animals
models of stress is very poorly recognized. Further more a role of
susceptibility to stress in modulatory effect of antidepressant drug on
tumour growth have not been studied. The aim of present study was
to establish the effect of individual reactivity to stress and prophylactic two weeks desipramine administration on metastatic colonization
of MADB 106 cells in lungs of Wistar rats. Rats were subjected by
three weeks to chronic mild stress (CMS) model of depression and
high-reactive and non-reactive rats were selected. Three months after
CMS termination high-reactive and non-reactive animals were further subdivided: for two additional weeks the rats received daily injection of desipramine or saline. Tumor cells were injected two hours
after last desipramine or saline administration. In stress-reactive,
vehicle treated rats increase of number of lung metastasis in comparison to stress non-reactive, vehicle treated rats were observed.
On the other hand chronic desipramine pretreatment significantly
increased survival rate and diminished number of lung metastasis in
stress-sensitive animals although did not show such effect in stressresistant animals. Increase in rate of animal survival and decrease in
lung metastasis can be connected with stimulatory effect of chronic
desipramine pretreatment on NK cell activity. This study was supported by grant N40109732/2074 from Poland’s MS and HE.
TVII.05
STRESS INDUCES CHANGES OF
CATECHOLAMINERGIC SYSTEM IN THE RAT SPLEEN
Laukova M.1, Bohacova V.1, Krizanova O.2, Kvetnansky R.1
1
Laboratory of Stress Research, Institute of Experimental
Endocrinology SAS, Bratislava, Slovak Republic; 2 Laboratory of
Biochemistry of Transport Systems, Institute of Molecular Physiology
and Genetics SAS, Bratislava, Slovak Republic
There is evidence that stress-induced activation of sympathoadrenal
system (SAS) affects the function of immune system. The aim of our
work was to investigate catecholamine (CA) levels, gene expression
of CA biosynthetic enzymes and adrenoceptors (ARs) in the spleen
of rats exposed to single and repeated (14×) immobilization stress
(IMO). Acute IMO did not affect adrenaline or noradrenaline (NA)
levels in the spleen. Repeated IMO induced a rise in both CAs suggesting increased CA biosynthesis. This stimulus was associated
with the increase in tyrosine hydroxylase (TH) protein, but with un-
358
detectable TH mRNA levels. That indicates predominantly neuronal
origin of this enzyme. Detection of dopamine-β-hydroxylase and
phenylethanolamine-N-methyltransferase gene expression points
to their local production and responsiveness to IMO. Stress differently affected individual types of splenic ARs. Exposure to acute
and chronic IMO increased β2-AR mRNA, which is the major immunomodulatory AR. Gene expression of α2A- and α2C-AR, the main
NA autoregulatory ARs, was reduced. As a consequence, NA level
increased. Thus, exposure to stress produces the rise of CAs in the
spleen. The elevation of β2-AR and a drop in α2-ARs mRNA, indicate their predominant function in immune system regulation, most
probably cytokine production via β2-AR and CA turnover via α2-AR
uptake as well as via CA biosynthetic enzymes activity. However, the
physiological role of this observation needs further investigation.
TVII.06
THE INFLUENCE OF AGING ON THE POPULATION OF
NGF CONTAINING NEURONS OF LIMBIC SYSTEM IN
THE RAT FOLLOWING OPEN FIELD STIMULATION
Badowska E., Cecot T., Klejbor I., Ludkiewicz B., Morys J.
Department of Anatomy and Neurobiology, Medical University
of Gdansk, Gdańsk, Poland
NGF (nerve growth factor) is involved not only in growth and survival of neurons but also promotes their age-dependent morphological changes (repair and remodeling) in normal life and during
stress. This study aimed to investigate an influence of ages, on
the changes of NGF immunoreactive (-ir) cells in the: amygdala,
hippocampus and hypothalamus caused by acute (one-time for 15
min) or repeated (21 days for 15 min daily) exposition to open field
(OF) test. Each group of age consisted of experimental and control
(non-stressed) Wistar male rats. To detected NGF-ir cells single
immunofluorescence staining was applied. Each control groups
revealed many of NGF-ir neurons in the studied structures. Following OF acute stimulation, the number of NGF-ir cells in all the
studied structures was higher in the three months old rats than that
of control ones; the level of NGF-ir cells in the one year old rats
was higher only in paraventricular nucleus of hypothalamus and in
central nucleus of amygdala. In two years old rats no changes was
observed in comparison with control animals. After OF repeated
exposition, the level of NGF-ir cells was similar to that observed
under acute one. These data demonstrated that the aging affected
the level of NGF-ir neurons caused by acute and repeated OF stimulation in the structures of limbic system. Stress duration did not
influence the level of NGF-ir neurons.
TVII.07
DEHYDROEPIANDROSTERONE SULPHATE (DHEAS)
IS REWARDING AND ALTERS THE REWARDING
EFFECT OF COCAINE IN THE CONDITIONED PLACE
PREFERENCE PROCEDURE (CPP)
Zajda M.1, Krzascik P.2, Majewska M.3
1
Departament of Neurology, 2 Departament of Clinical and
Experimental Pharmacology, Medical University of Warsaw,
Warszawa, Poland; 3 Department of Physiology and Pharmacology
of the Central Nervous System, Institute of Psychiatry and Neurology,
Warszawa, Poland
DHEAS is endogenous allosteric antagonists of the GABA(A)
receptor and has other direct and indirect actions in CNS. It
is metabolized to other neuroactive steroids. Prior studies revealed an ambiguous role of DHEA(S) in cocaine dependence,
suggesting interactions with cocaine effects in the brain. This
preclinical study was undertaken to evaluate direct influence of DHEAS on the rewarding effects of cocaine, using
the CPP test in rats. Male Wistar rats (250–300 g) were used.
The conditioned place preference experiment consisted of preconditioning conditioning and post-conditioning phases. Drug
injections were done i.p. only during the conditioning phase.
Cocaine had a biphasic dose-dependent rewarding effect (inverted U type). The doses 5 and 10 mg/kg were clearly rewarding, as they increased rats’ preference for cocaine injection
site, while at the dose 20 mg/kg, cocaine rewarding effect was
lost. DHEAS alone had a similar biphasic rewarding effect (the
effect of 40 mg/kg was maximal and statistically significant).
Pretreatment of rats with 40 mg/kg of DHEAS before injection
of cocaine noticeably changed its rewarding effect, shifting cocaine dose response curve to the left. DHEAS, per se has rewarding properties and it enhances the rewarding effects of low
doses of cocaine, but decreases the rewarding effect of higher
doses of cocaine. Funded by EC grant MEXC-CT-2006-42371
to M.D. Majewska.
TVII.08
ANDROSTERONE HAS REWARDING AND AVERSIVE
PROSPERITIES AND ALTERS THE REWARDING
EFFECT OF COCAINE IN THE CONDITIONED PLACE
PREFERENCE PROCEDURE (CPP)
Zajda M.1, Krzascik P.2, Majewska M.3
1
Departament of Neurology, 2 Departament of Clinical and
Experimental Pharmacology, Medical University of Warsaw,
Warszawa, Poland; 3 Department of Physiology and Pharmacology
of the Central Nervous System, Institute of Psychiatry and Neurology,
Warszawa, Poland
Our previous study showed that the neurosteroid dehydroepiandrosterone sulfate (DHEAS) alters the rewarding effects of
cocaine. Because DHEAS is metabolized to androsterone, which
has opposite synaptic activity, we wondered about contribution
of androsterone to the above phenomenon. Here we examined
androsterone’s effect on the rewarding properties of cocaine in
CPP test. Male Wistar rats (250–300 g) were used. The CPP procedure consisted of pre-conditioning, conditioning and post-conditioning phases. Drug injections were done i.p. Androsterone
alone was rewarding at doses 1 mg/kg and 10 mg/kg, while the
dose of 40mg/kg was visibly aversive. Cocaine had a biphasic
dose-dependent rewarding effect (inverted U type): the doses 5
and 10 mg/kg were rewarding, while at dose 20 mg/kg, cocaine
rewarding effect was lost. Pretreatment with 1mg/kg of androsterone increased the rewarding effect of all cocaine doses, especially the dose of 20 mg/kg, but the rewarding dose of 10 mg/
kg of androsterone had no effect on the rewarding prosperities
of cocaine. All doses of cocaine decreased the aversive effect of
40 mg/kg of androsterone. Androsterone per se has a biphasic
rewarding-aversive effects in the CPP test and influences the rewarding properties of cocaine, which might be explained by two
mechanisms: potentiation of reward at low doses and reduction of
the aversive effect of the high doses. Funded by EC grant MEXCCT-2006-42371 to M.D. Majewska.
TVII.09
INTERACTIONS OF NEUROSTEROIDS –
DEHYDROEPIANDROSTERONE SULPHATE (DHEAS)
AND ANDROSTERONE - WITH COCAINE IN
BEHAVIOURAL DESPAIR TEST (PORSOLT) IN RATS
Krzascik P.1, Zajda M.2, Majewska M.3
1
Department of Clinical and Experimental Pharmacology, 2
Department of Neurology, Medical University of Warsaw, Warszawa,
Poland; 3 Department of Physiology and Pharmacology of the Central
Nervous System, Institute of Psychiatry and Neurology, Warszawa,
Poland
Previous studies showed that high plasma levels of endogenous
DHEAS correlated with resiliency and ability of cocaine addicts
to maintain prolonged abstinence after the therapy (Wilkins et al.
2005), while addicts treated with high dose of exogenous DHEA
used more cocaine that those administered placebo (Shoptaw et al.
2004). Such ambiguous outcomes could be due to mixed actions
of DHEAS and its metabolite, androsterone, which have opposite
neuronal activities. Here we examined acute effects of both neurosteroids in Porsolt test and their interactions with 5 mg/kg of cocaine – the dose reported to have antidepressive action, but which
does not influence locomotor activity in the open field. Wistar rats
(250-300 g) were used. All substances were injected i.p.; steroids
30 min and cocaine 5 minutes before the test, which measures locomotor activity of rats in narrow water containers. Cocaine at
dose 5 mg/kg significantly increased rats’ locomotor activity in
the Porsolt test. Neither DHEAS (1, 5, 10, 40, 80 mg/kg) nor androsterone (0,1, 1, 10, 40 mg/kg) per se had any effect on activity
in this test. DHEAS dose-dependently potentiated the effect of cocaine, but androsterone had no influence on this cocaine action.
Acutely administered DHEAS appears to increase antidepressive
effects of cocaine, but androsterone has no such influence. Funded
by EC grant MEXC-CT-2006-42371 to M.D. Majewska.
peripheral circulation require intact midcervical vagi. Supranodose
vagotomy failed to eliminate the hypertension evoked by arvanil.
TVII.11
ROLE OF CHEMOSENSITIVE RTN NEURONS IN
MEDITATING RESPIRATORY ACTIVATION AFTER
HYPOTHALAMIC STIMULATION
Fortuna M., Stornetta R., West G., Guyenet P.
Department of Pharmacology, University of Virginia, Charlottesville,
VA, USA
Stimulation of the dorsomedial nucleus (DMH) or the perifornical
area (PeF) of the hypothalamus activates breathing. The descending neuronal pathways that mediate this response are unknown. In
the present experiments we test the possibility that this pathway
includes a relay in the retrotrapezoid nucleus (RTN). The RTN
resides at the rostral end of the medulla oblongata and consists of
intrinsically pH-sensitive glutamatergic neurons that are proposed
to contribute to CO2 stabilization by regulating the activity of the
Central Respiratory Pattern Generator (CPG). The experiments
were done in adult isoflurane-anesthetized rats. Stimulation of
DMH/PeF with the selective GABA A receptor antagonist gabazine
increased phrenic nerve activity and the discharge rate of simultaneously recorded RTN units. This upregulation was observed
at all levels of end-expiratory CO2. After silencing the CPG with
intravenous morphine (10–15 mg/kg), hypothalamic stimulation
was still capable of enhancing RTN activity. In addition, both
DMH/PeF stimulation or hypercapnia greatly increased the number of RTN neurons that expressed Fos when compared to rats
maintained under control hypocapnic conditions. We conclude
that the DMH/PeF region of the hypothalamus controls respiration at least in part by regulating the activity of the retrotrapezoid
nucleus. Support: HL74011, HL 28785.
TVII.10
ROLE OF THE AFFERENT VAGAL PATHWAY IN
MODELING OF CARDIO-RESPIRATORY RESPONSE
TO ARVANIL IN ANAESTHETIZED RATS
Kopczynska B.
Laboratory of Respiratory Reflexes, Mossakowski Medical Research
TVII.12
BINGE ALCOHOL EXPOSURE DURING PUBERTY
INCREASES THE EXPRESSION OF GENES INVOLVED
IN STRESS RESPONSES
Przybycien M.1, Rao Y.2, Pak T.3
1
Neuroscience Program, 2 Molecular Biochemistry Program,
3
Department of Cell Biology, Neurobiology and Anatomy, Loyola
University Medical Center, Chicago, IL, USA
Arvanil is metabolically stable hybrid between anandamide and
capsaicin and an agonist of cannabinoid CB1 and vanilloid VR1
receptors. Arvanil is able to alleviate hyperkinesia typical in rat
model of Huntington’s disease, spasticity, pain, tremor and other
signs of disease in rat model of multiple sclerosis. The drug reveals
anti-tumour and anti-inflammatory action. The present study was
designed to test the role of the vagal pathway in post-arvanil cardiorespiratory response. Cardio-respiratory effects of an intravenous
injection of arvanil were investigated in 21 urethane-chloralose anaesthetised and spontaneously breathing rats. Bolus injection of 0.8
mg kg-1 of arvanil into the right femoral vein induced in all neurally
intact rats a significant increase of tidal volume (VT) and diaphragm
activity as well as hypertension coupled with fall in respiratory rate
(f). Bilateral midcervical vagotomy precluded the alteration of respiratory parameters without any changes in cardiovascular effects.
Arvanil-induced increase in mean arterial blood pressure (MAP)
still persisted even after supranodose vagotomy. Results indicated
that the respiratory effects evoked by arvanil administered via the
Teenage alcohol abuse is a fundamental health concern and it can
have permanent effects on brain function. We identified the effects
of adolescent binge exposure on regulators of stress and anxiety
responses: corticotrophin-releasing hormone (CRH) and arginine
vasopressin (AVP). We hypothesized that binge ethanol exposure
during puberty increases the expression of these genes in the paraventricular nucleus (PVN) of the hypothalamus. Animals wew
handled daily for 7 days and then divided into 4 groups (n=6/8): (1)
untreated, (2) intraperitoneal (i.p.) saline injection (for 8 days), (3)
acute ethanol exposure (saline for 7 days and ethanol (3 g/kg) on
the last day) and (4) binge ethanol exposure (3 days of ip ethanol
injection, 2 days of saline and 3 days of ethanol). Treatments started
at PND37 and animals were sacrificed at PND44 1 hour after the
last injection; trunk blood was collected, brains removed, and rapidly frozen. Blood alcohol level reached 153 ± 12 mg/dl in males
and 187 ± 23 mg/dl in females. Both acute and binge alcohol treatments increased CORT levels. Brains were sectioned at 200 μm on
a freezing microtome and the PVN was microdissected. Total RNA
360
was isolated from the PVN and quantitative real time RT-PCR was
performed. In the PVN binge ethanol exposure increased CRH
and AVP gene expression (P<0.05) suggesting that it might have
profound effects on the development of the hypothalamo-pituitaryadrenal axis and could increase the risk of adult anxiety disorders.
TVII.13
MATERNAL SEPARATION AFFECTS TYROSINE
HYDROXYLASE AND NCAM EXPRESSION IN
DOPAMINERGIC STRUCTURES OF JUVENILE,
ADOLESCENT AND ADULT RAT BRAIN
Chocyk A., Mackowiak M., Dudys D., Przyborowska A.,
Wedzony K.
Laboratory of Pharmacology and Brain Biostructure, Institute
of Pharmacology PAS, Kraków, Poland
Stressful experiences in early life can influence brain development
and increase the risk for mental disorders. However the specific impact of early life stress on maturation of dopaminergic (DA) system
is still unknown. We applied maternal separation (MS) paradigm
in rats, on postnatal (P) days 1–14, to investigate its impact on expression of tyrosine hydroxylase (TH) and NCAM proteins in DA
structures of juvenile (P15), adolescent (P35) and adult (P70) rat
brain. On P15 MS decreased the number of TH+ neurons in the substantia nigra (SN) pars compacta of females as well as in the SN
pars reticulata (SNr) and the ventral tegmental area (VTA) of both
females and males. The effect persisted until P35 only in the VTA of
MS females. Oppositely on P70, an increase in the number of TH+
neurons was observed in the VTA of females. Additionally on P35
a transient rise in TH+ cells was detected in the SNr of MS females.
MS did not change the expression of TH in the striatum, nucleus accumbens and medial prefrontal cortex (mPFC). On P15 a decrease
in the level of 140 kDa NCAM was observed in the SN and striatum
of MS females and a decrease in 180 kDa NCAM in the VTA of MS
males. On P35 MS diminished the expression of 180 kDa NCAM in
the SN and mPFC of females. Finally, on P70 a decrease in 180 kDa
NCAM was observed in the mPFC of MS males. The results of the
present study strongly indicate that early life stress can affect maturation and plasticity of DA system at different life stages.
TVII.14
COMBINED ACTION OF VIBRATION AND LOWINTENSITY ELECTROMAGNETIC RADIATION
ON SPIKE ACTIVITY OF SUPRAOPTIC CELLS
Grigoryan G., Minassian S., Sahakyan S.
Department of Human and Animal Physiology, Yerevan State
University, Yerevan, Armenia
Spontaneous spike activity of supraoptic cells of hypothalamus
after 30-day-long exposure of low-intensity extremely-high frequency radiation (42.2 GHz, 0.19 mW/cm 2, daily for an hour) on
vibration-stimulated rats (60 Hz, 0.4 mm, 2 hours daily) was analyzed. The distributions of registered cells by the degree of regularity of interspike intervals (ISI), dynamics of succession of ISI
and discrete frequency ranges, as well as the mean spike frequency and the coefficient of variation of ISIs in naive control and after treatment were evaluated. 30-day-long exposure was followed
by significant (P<0.05/P<0.01) shifts in distribution by degree of
regularity and dynamics of succession of ISI. These changes were
significant in comparison with isolated influence of vibration. The
significant decrease of mean spike frequency up to 13.5 ± 1.7 imp/s
(P<0.01) was revealed. It was accompanied by reliable (P<0.01)
abrupt increase of quantity of low-frequency cells as well as decrease of numbers of high- and medium-frequency units. These
changes were statistically insignificant in comparison with data
from isolated action of vibration. The coefficient of variation of
ISIs after treatment was increased up to 105.3 ± 4.3% (P<0.05).
Thus, we suppose that the effect of low-intensity extremely-high
frequency radiation depends on initial state of organism.
TVII.15
TIME-DEPENDENT EFFECTS OF SWIM STRESS ON
EARLY-LTP IN THE HIPPOCAMPAL CA1 IN FREELY
MOVING RATS
Yeritsyan N., Frey J.
Department of Neurophysiology, Leibniz Institute of Neurobiology,
Center of Learning and Memory Research, Magdeburg, Germany
Stress has been well-documented to affect hippocampal long-term
potentiation (LTP), which is widely believed to underlie learning
and memory at the cellular level. However, only few reports addressed the issue regarding the timing of presenting a stress episode in relation to LTP induction. Previously we could show that
in the dentate gyrus a protein synthesis-independent early-LTP can
be reinforced by swim stress into a protein synthesis-dependent
late-LTP when the stress episode was applied within a time window of about 30 min around tetanization (Korz and Frey 2003).
Considering the important role that the hippocampal CA1 plays in
the processing of spatial and temporal information, we were now
interested in studying the influence of swim stress on functional
plasticity events in the CA1 region. Field potentials were recorded
in freely moving rats subjected to a brief episode of swim 15 min
before or after early-LTP induction in the CA1 by stimulating the
contralateral CA3. Our results revealed that a swim episode alone
exerted a transient depressing effect on baseline values of recorded
field potentials. Swim stress presented shortly after the induction of
early-LTP resulted in its depotentiation. Furthermore, if the same
stress protocol preceded early-LTP induction, it prevented the potentiation and induced a long-lasting depression of field potentials.
These data suggest that swim stress differentially alters synaptic
plasticity in the CA1 when compared with the dentate gyrus.
TVII.16
c-Fos, 5HT AND CRF IMMUNOSTAINING OF THE
PREFRONTAL CORTEX AREAS THE IN THE RATS
DIFFERING IN THE STRENGTH OF A FEAR RESPONSE
Wislowska-Stanek A.1, Lehner M.2, Taracha E.2, Skorzewska A.2,
Maciejak P.1,2, Szyndler J.1, Plaznik A.1,2
1
Medical University of Warsaw,Warszawa, Poland; 2 Department of
Neurochemistry, Instituite of Psychiatry and Neurology, Warszawa,
Poland
The aim of the study was to examine the neurochemical background of differences in the individual responses to conditioned
aversive stimuli, using the strength of a rat conditioned freezing
response (the contextual fear test), as a discriminating variable.
It was shown that high responder (HR), i.e. rats with duration of
a freezing response one standard error, or more, above the mean
value, had a higher c-Fos activity in the FrA and PrL prefrontal
cortical areas, and stronger 5HT immunostaining in the FrA.
However, these animals had lower CRF immunostaining in the
same cortical areas in comparison with low responder (LR), i.e.
rats with the duration of a freezing response one standard error, or
more, below the mean value. The LR group vocalized more during
test session in the aversive band, and had higher serum levels of
corticosterone, examined 10 min after test session. It was shown
that different natural patterns of responding to conditioned aversive stimuli are associated with different expression of CRF and
serotonergic- innervation of prefrontal cortical areas.
TVII.17
THE INFLUENCE OF CRF AND α-HELICAL CRF(9-41)
ON RAT FEAR RESPONSES AND AMINO ACIDS
CONCENTRATION IN THE CENTRAL NUCLEUS
OF AMYGDALA
Skorzewska A.1, Bidzinski A.1, Hamed A.2, Lehner M.1,
Turzynska D.1, Sobolewska A.1, Szyndler J.1, Maciejak P.1,2
1
Department of Neurochemistry, Instituite of Psychiatry and
Clinical Pharmacology, Medical University of Warsaw,Warszawa,
Poland
In the previous study we showed that exogenous CRF (1 μg/rat, icv)
enhanced rat-freezing responses and increased expression of CRFrelated immunoreactive complexes in the amygdala. On the other
hand, a non-selective CRF receptor antagonist, α-helical CRF(9-41)
(10 μg/rat, i.c.v.) decreased rat fear response and increased the concentration of GABA in the amygdala (in vitro). The aim of the present study was to examine the effects of CRF and α-helical CRF(9-41),
on the freezing reaction in the conditioned fear test and the release
of amino acids in the central nucleus of amygdala (CeA), using microdialysis technique. It was found that CRF increased the concentration of aspartate, glutamate and Glu/GABA ratio in the CeA,
an effect that preceded an increased expression of anxiety-like responses, which appeared 15 min after drug administration. α-helical
CRF(9-41), decreased rat freezing responses and increased the local
concentration of GABA, during the first 30 min of observation. The
present data show an important role of CRF in the central nucleus of
the amygdala in the integration of anxiety-related, biochemical and
behavioural responses, and suggest an involvement of amino acids
innervation of the CeA, in the effects of this neurohormone.
TVII.18
THE POWER OF FREQUENCY BANDS IN ELECTRICAL
ACTIVITY OF LIMBIC STRUCTURES OF THE ROMAN
HIGH AND LOW AVOIDANCE RATS DIFFERS IN
STRESS-DEPENDENT MANNER
Meyza K., Boguszewski P., Olszewski M., Kasicki S.,
Zagrodzka J.
Biology PAS, Warsaw, Warszawa, Poland
The psychogenetically selected Roman High (RHA/Verh) and Roman Low (RLA/Verh) Avoidance rats constitute a widely accepted
model of diverse emotional reactivity. They show divergent stressrelated behavioral and neuronal responses when confronted with a
novel and/or stressogenic environment (Meyza et al. 2009). Here, we
have explored the possibility that the difference might be also reflected in the electrical activity of prefrontal cortex (mPFC), amygdala
(Amy) and hippocampus (CA1) – structures involved in generation
of emotions. Spectral analysis of telemetrically recorded local field
potentials (LFPs) from mPFC, Amy and CA1 in freely moving rats
performing behavioral tests differing with aversiveness (Open Field,
Elevated Plus Maze, Hole Board test and Acute Restraint) showed
that power of 4 distinct frequency bands (delta: 0–3 Hz, theta: 4–12
Hz, beta: 13–30 Hz and gamma: 31–90 Hz) is higher in the LFPs of
RLA/Verh than of RHA/Verh rats, especially in the CA1 and mPFC.
This difference seems to be stress-dependent (among tests measuring spontaneous behavior). Acute restraint elicited differences only
in the delta and gamma bands in the mPFC. Strikingly, the difference is most clear while the animal is performing a decisive/risk
assesment behavior (e.g. leaning towards aversive part of the arena).
Moreover, individual differences in the power of bands can be observed at that time, thus they may be considered a neuronal correlate
of individual differences in emotional reactivity.
TVII.19
SEROTONIN AND CORTICOSTERONE –
NEUROCHEMICAL INDICATORS OF STRESS
Koprowska M., Krotewicz M., Strzelczuk M.
Laboratory of Neurophysiology, University of Lodz, Łódź, Poland
The anxiety tests are widely used to study behaviour of rats in the
stress-inducing situation. The study presents a comparative analysis
of the influence of different stressogenic stimuli on the serotonergic
activity in the emotional brain regions and on plasma corticosterone
concentration in rats. The animals were subject to two various behavioral tests, and subsequently the concentration of serotonin (5HT), its metabolite 5 – hydroxyindoleacetic acid (5-HIAA) and of
plasma corticosterone concentration were determined using highperformance liquid chromatography with electrochemical detection
(HPLC-ED). Stressogenic stimuli in the tests performed were: light
of high volume (light dark transition test – LDT) or open field (open
field test – OF). HPLC-ED analysis showed a significant increased
of 5-HT level in the amygdala (AM) after using both tests. HPLCED analysis showed also an increased of 5-HIAA concentration in
the frontal cortex (CTX), in the hippocampus (HI), in the AM after
using the LDT-test and an increased of 5-HIAA concentration in
the CTX and in the AM after using OF-test. Simultaneously, the
analysis showed elevation of corticosterone concentration in rats
exposed to LDT and OF. These results confirm that serotonin and
coricosterone participate in the regulation of emotional response
and they are important neurochemical indicators of stress.
TVII.20
THE ROLE OF CLONIDINE IN BEHAVIORAL AND
CORTICOSTERONE RESPONSES TO SOCIAL
COMPETITION
Krotewicz M., Koprowska M., Strzelczuk M.
It is postulated that the noradrenergic brain neuronal activity is
closely related to the control of the hypothalamo-pituitary-adrenocortical (HPA) system. Our previous observations showed that
the brain noradrenergic system is implicated in the control of social position studied in social competition tests. In order to obtain
some further evidence speaking for a specific role of noradrenaline
system activity in the control of social position we studied the effects of administration of the α2-adrenoceptor agonist clonidine on
social behavior analyzed in the social competition test. Simultaneously, the concentration of corticosterone (RIA) was measured in
the plasma of rats after social competition test and tested individually. We used an acute social competition situation that consisted
362
of simultaneous placing of two experimental rats into the familiar
cage supplied with feeder of glucose pellets. The obtained data revealed that social competitive behavior was affected by peripherally administered clonidine. The animals got better position for competition and received higher social rank in pairs after stimulation of
α2-drenoceptor. Plasma corticosterone concentration was elevated
in rats injected peripherally with clonidine in comparison with individually tested rats. The obtained results suggest that stimulation
of α2-adrenoceptor affects HPA system activity and may facilitate
appearance of tendency to social confrontations in rats.
TVII.21
THE EFFECT OF THE AUTONOMIC NERVOUS SYSTEM
ON THE SURVIVAL OF RATS IMPLANTED WITH
YOSHIDA ASCITES TUMOR CELLS
Ondicova K.1, Lackovicova L.2, Valaskova Z.1, Macikova I.3,
Perzelova A.3, Hulin I.1, Gidron Y.4, Mravec B.1
1
Institute of Pathophysiology, Faculty of Medicine, Comenius
University, Bratislava, Slovak Republic; 2 Institute of Experimental
Endocrinology SAS, Bratislava, Slovak Republic; 3 Institute of
Anatomy, Faculty of Medicine, Comenius University, Bratislava,
Slovak Republic; 4 School of Health Sciences and Social Care, Brunel
University, London, UK
Even if it is suggested that stress does not directly induce cancer
there is plenty of evidence that shows that stress represents an important factor facilitating cancer progression, however the exact
mechanisms and pathways are not known in details. Because the
autonomic nervous system plays an important role in the elaboration of stress response, we investigated the effect of the disruption
of the sympathetic or parasympathetic system on the survival of tumor bearing rats. We used male Wistar rats in which we performed
either chemical sympathectomy induced by intraperitoneal application of 6-OH dopamine or subdiaphragmatic vagotomy by surgical dissection of the vagus nerve. After a regeneration period we
administered intraperitoneally to sympathectomized, vagotomized
and sham operated rats Yoshida ascites cells. We have found that
whereas chemical sympathectomy significantly reduced the survival of tumor bearing rats, subdiaphragmatic vagotomy had only a
slight effect on reducing the survival of rats implanted by Yoshida
cells. Our findings suggest that the autonomic nervous system,
especially its sympathetic division, plays an important role in the
regulation of the development of Yoshida ascites tumor cells in rats.
We suggest that whereas long lasting sympathetic activation as a
consequence of exposure to chronic stress might have a promoting
effect on cancer growth, the sympathetic system might have, during
basal conditions, a modulatory effect on tumor progression.
TVII.22
RELAXIN-3 INNERVATION OF THE
INTERGENICULATE LEAFLET IN THE RAT
Blasiak A.1, Blasiak T.1, Czubak W.1, Gundlach A.2,
Lewandowski M.1
1
University, Kraków, Poland; 2 Florey Neuroscience Institutes, The
University of Melbourne, Melbourne, Australia
The intergeniculate leaflet of the thalamus (IGL), a subdivision of
the lateral geniculate body, is a component of the biological clock,
responsible for, amongst other things, the synchronizing effect of
timed caloric restriction on locomotor activity in rodents. The IGL
has a distinct functional and anatomical profile amongst thalamic
nuclei, including strong peptidergic inputs (e.g. orexin and PACAP). Recently a dense network of terminals immunoreactive for relaxin-3 (RLX3) was identified in IGL. RLX3 is a newly discovered
neuropeptide expressed in high levels in brain. The physiological
actions of RLX3 are largely unknown, but recent studies have suggested influences on feeding, stress responses and spatial memory.
The majority of RLX3 neurons are located in the nucleus incertus
(NI), but prior anatomical studies have excluded the NI as a source
of the RLX3 in the IGL. In this study, we used neural tract-tracing
and immunohistochemistry to demonstrate that the major source of
RLX3 fibers within IGL originates from RLX3 neuron populations
in the periventricular- and periaqueductal gray. Furthermore, some
of these IGL-projecting RLX3 neurons express corticotropin-releasing factor receptors, indicating that the RLX3 innervation may
transmit stress-related responses to the IGL. Since stress is one of
the non-photic factors capable of synchronizing behavioural rhythmicity, our data suggest that RLX3 might be another key element
in the complex mammalian circadian system.
TVII.23
ANALYSIS OF THE INTENSITY OF ANXIETY
RESPONSE MEASURED WITH ELEVATED PLUS-MAZE
TEST
Strzelczuk M., Koprowska M., Krotewicz M.
Anxiety plays undoubtedly important role in the life of the individual units and leads to the search for better mechanisms for adaptation engaged in mobilizing for action. An important role in
the regulation mechanisms for the escape and providing increased
activity of the organisms plays catecholamines. The neurochemical
background of anxiety is increase of concentration of noradrenaline
and dopamine in the emotional structures of the brain. In the present work we used Elevated Plus-Maze test (EPM test). The EPM
test is widely used to study behavior of rodents for exploration and
emotionality. It was made an analysis of changes in concentration
of noradreneline (NA), dopamine (DA) and their metabolites in selected brain regions in rats after the EPM. HPLC analysis showed
elevation of MHPG level in the hypothalamus (HPT) and DOPAC
in the frontal cortex (CTX) of rats. These results indicate stimulation of the dopaminergic system activity in the CTX with simultaneous increase of the noradrenergic system activity in the HPT.
TVII.24
EFFECTS OF MATERNAL DEPRIVATION ON
BEHAVIOR AND BRAIN DEVELOPMENT IN ADULT
LIFE OF MICE
Gajerska M., Glowacz A., Turlejski K., Djavadian R.
Maternal deprivation model was used for studying the influence of
perinatal stress on the behavior of adult mice. Swiss mice pups were
separated from their mothers for two hours daily from postnatal day 1
(P1) to P7. One group of pups has been treated with buspirone immediately before each period of maternal separation (P1–P7). At the age
of 1–3 months mice were subjected to a battery of behavioral tests.
Then they were sacrificed, their brains were removed and used for
immunohistochemistry staining or immunoblotting (Western blot)
with antibodies directed against glucocorticoid or mineralocorticoid
receptors. We found that in the open field test and the plus maze test
the stressed mice showed greater anxiety than non-stressed animals.
Anxiety in stressed mice treated with buspirone was lower than in
stressed only mice. Next, mice were tested either in a water maze
test or an object recognition test where their memory and learning
performance was assessed. We did not find any significant differences between groups in these two tests. These results demonstrate
that stressing early life events, like maternal separation, permanently
disturb emotionality of the adult mice but do not affect their learning
or memory functions. Deleterious effects of stress may be prevented
by pharmacological treatment. Supported by the Polish Ministry of
Science and Higher Education grant No 3757/B/P01/2007/33.
TVII.25
ELECTRICAL STIMULATION OF THE MEDIAL
SEPTAL NUCLEUS INCREASES CYTOTOXIC
ACTIVITY OF NK CELLS IN THE SPLEEN AND
PERIPHERAL BLOOD IN RATS
Myslinska D., Glac W., Badtke P., Grembecka B., Plucinska K.,
Wrona D.
Poland
The medial septal nucleus (MS) is a forebrain limbic structure involved in learning and memory mechanisms. In previous study we
found that electrolytic lesion of the medial septal nucleus caused
depression of the peripheral blood natural killer cell cytotoxicity
(NKCC) and the leukocyte number. In the sham operated group
mere insertion of electrodes into the MS evoked transient NKCC
enhancement, probably resulting from mechanical MS stimulation.
To check this effect in the present study, we evaluated both spleen
and blood NKCC (51Cr-release assay) and large granular lymphocytes (LGL) number (a morphological method) after chronic electrical stimulation (constant current 0.1 ms duration cathodal pulses
delivered at a frequency of 50 Hz during 30-min daily session for 14
consecutive days) of MS in conscious, freely moving rats (n=12). Additionally, peripheral blood leukocyte, lymphocyte and neutrophile
number was mesaured. Chronic MS stimulation caused significant
blood NKCC augmentation and LGL number (25.86 ± 9.31% vs.
15.75 ± 4.75%, P<0.01) in comparison to the sham group (n=13). No
significant effect was found in the spleen (27.14 ± 9.99% vs. 28.58
± 8.04%). A week after termination of the stimulation procedure all
measured parameters returned to the baseline. The results obtained
indicate that such limbic structure as medial septum enhances antitumor and antiviral function and number of NK cells.
TVII.26
UNILATERAL DAMAGE TO THE NUCLEUS
ACCUMBENS DECREASES PERIPHERAL BLOOD NK
CELL NUMBER IN RATS
Plucinska K., Grembecka B., Glac W., Badtke P.,
Jerzemowska G., Myslinska D., Orlikowska A., Wrona D.
Poland
The nucleus accumbens (Acb), which is major terminal fields for
the dopamine fibers originating in the ventral tegmental area (VTA)
plays a pivotal role in the integration of signals arising from the
limbic and cortical areas that mediate the motivational, homeostatic
and motor aspects of behaviour. In the present work, we assessed
the effects of unilateral electrolytic lesion of the Acb on peripheral
blood lymphocyte number in freely moving rats. Before unilateral
Acb lesion, all rats (n=10) were exposed to chronic (14 consecutive
days) VTA electrical stimulation to obtain feeding or exploration
response and the baseline level of lymphocyte numbers. On the 2nd
day after electrolytic lesion of Acb (2 mA, 15 s) the percentage of
blood T, B and NK lymphocytes, TCD3+CD4+ and TCD3+CD8 subsets were determined by flow cytometry. As compared to the baseline, the significant decrease in NK cell percentage (51.10 ± 14.05%
vs. 20.3 ± 5.84%, P<0.01) following Acb lesion was observed. In
contrast, there were no significant differences in the percentage
number of B (17.7 ± 2.38% vs. 22.25 ± 5.28%) and T (39.31 ± 9.75%
vs. 42.10 ± 7.21%) cells between the baseline and post-lesion values.
Accordingly, post-lesion values of T helper and T cytotoxic subset
were as follow: 30.35 ± 5.31%, 8.02 ± 1.11%. These findings suggest that Acb, which is partially responsible for motivational states,
plays a crucial role in innate antitumor and antiviral response.
TVII.27
CHANGES OF BRAIN-DERIVED NEUROTROPHIC
FACTOR (BDNF) IMMUNOREACTIVE CELLS NUMBER
IN HYPOTHALAMIC NUCLEI AFTER CHRONIC
STRESS IN YOUNG AND AGED RATS
Cecot T., Badowska E., Klejbor I., Domaradzka-Pytel B.,
Morys J.
Department of Anatomy and Neurobiology, Medical University
of Gdansk, Gdańsk, Poland
It is postulated, that brain-derived neurotrophic factor (BDNF) have
been implicated in the neurobiological mechanisms underlying
brain plasticity after chronic stress. The objective of this study was
to evaluate influence of chronic stress on brain plasticity measured
by BDNF immunoreactivity in brain structures of young (P28) and
adult (P360) rats. 26 male Wistar rats were exposed to 15 min daily
open field (OF) or forced swim test (FS) during three weeks. Fluorescent immunohistochemistry was used to localize BDNF positive
cells in hypothalamic areas connected with stress response: both
parvo- and magnocellular divisions of the paraventricular nucleus
(PVp and PVm) and the supraoptic nucleus (SO). In animals aged P28
chronic OF i FS stress caused a statistically significant (P<0.001) decline in the number of BDNF-ir cells in both parts of the PV and SO.
In contrast, in rats P360 was not observed any change in the number
of BDNF-ir cells after chronic OF stimulation compared to control
in PVp and SO. In summary: age of rats subjected to chronic stimulation OF FS or stress had an impact on changes in the number of
BDNF-ir cells in the tested hypothalamic nuclei.
TVII.28
GAP JUNCTION IN THE HIPPOCAMPAL FORMATION
NETWORK: INTERACTION BETWEEN GAP JUNCTION
BLOCKER AND OPENER
Bocian R., Posluszny A., Kowalczyk T., Golebiewski H.,
Konopacki J.
Department of Neurobiology, University of Lodz, Łódź, Poland
Gap junction (GJ) channels provide an aqueous pathway for the passage ions current and small molecules between cell cytoplasms. It
has been demonstrated that various physiological stimuli, experimental treatments and chemical agents can modulate activity of
those structures. In our previous studies we showed that intrahippocampal injection of carbenoxolone (GJ blocker) abolished local recorded theta rhythm in anesthetized rats. However, intrahippocam-
364
pal formation (HPC) administration of trimethylamine (GJ opener)
evoked an opposite effect i.e. well synchronized theta activity. The
similar findings were also obtained in many experiments carried out
on awake animals and in studies conducted in in vitro conditions.
In the present studies, performed on anesthetized rats, the interaction between GJ blocker and opener, separately injected into HPC on
locally registered theta rhythm, were established. When diminishing effect of HPC injection of carbenoxolone (100 μg/1 μl) on theta
rhythm amplitude and power was observed, trimethylamine (30 μg/1
μl) was locally administered. Trimethylamine was found to alleviate
blocking effect on spontaneous HPC theta rhythm. In the next experiment, all rats pretreated with trimethylamine were injected with
carbenoxolone. GJ blocker was found to prevent facilitating effect
of trimethylamine on theta rhythm. The mechanism underlying GJ
involvement in theta rhythm generation in vivo is discussed. These
studies were supported by grant MNiSzW NN 401 2811 33.
ergic agonist, carbachol produce theta rhythm in HPC slices in vitro.
The evaluation of theta amplitude and phase profile indicates the existence of two theta generators located in CA1 and DG areas. Moreover,
model of slice preparation demonstrated that in the CA3c HPC region
the third RSA generator is localized. In our previous experiments
we hypothesized that completely isolated DG area is not capable of
theta rhythm generation. In the present study we attempted to verify
this hypothesis, using model of combined transected HPC slices. We
analyzed theta activity in two different transected slices: DG slices
containing CA1 area and DG slices containing CA3c region. We demonstrated that both CA1 and CA3c regions are capable of independent generation high amplitude RSA, whereas theta recorded in DG
was characterized by significantly lower theta amplitude. Our in vitro
obtained findings are consistent with earlier reports suggesting that
HPC theta generators are localized in CA1 and CA3c but not in DG
region. Supported by grant MNiSzW NN 303 091 834.
TVII.29
THE EFFECT OF CARBACHOL ON HIPPOCAMPAL
THETA IN CARBENOXOLONE PRETREATED RATS
Posluszny A., Bocian R., Kowalczyk T., Golebiewski H.,
Konopacki J.
TVII.31
DISCHARGE PATTERNS OF THETA-RELATED CELLS
IN TRANSECTED HIPPOCAMPAL FORMATION SLICES
Kowalczyk T., Golebiewski H., Bocian R., Posluszny A.,
Konopacki J.
Among the number of the mechanisms of signal transmission evolved
in biological systems, a distinctive feature of gap-junction coupling
is ability of direct high-speed information spreading over the space
of coupled cells. It is performed on cytoplasmic tracks provided by
the channel structure of gap junction assemblies (electrical synapses). In the nervous system this high-speed pathway is considered as
a promoter of neuronal synchrony, and so – an underground for oscillatory events. On the other hand, it is well known that to produce
oscillations, the brain tissue needs qualitatively and quantitatively
balanced neurotransmitter input feeding the oscillation-generating
networks. The question arise what is the real contribution of the two
signalling systems: electrical and chemical to oscillatory activity.
Hippocampal formation (HPC) theta rhythm is one of the best synchronized brain oscillations. Our earlier experiments demonstrted
that the cessation of gap junction coupling had a spectacular effect
on HPC spontaneous theta rhythm. HPC administrations of carbenoxolone (CBX), a gap junction blocker, produced progressive
impairment of theta oscillations up to their abolition. In order to estimate if high-level enhancement of chemical transmission is capable
of counteracting the gap junction blockage, in the present work carbachol, a cholinergic agonist, was injected into CBX pretreated hippocampus. Under CBX-blockage no effect of carbachol was noticed.
Grant MNiSzW NN 401 2811 33.
Hippocampal formation (HPC) theta rhythm is approximately sinusoidal, most prominent and well-documented EEG pattern recorded
from the mammalian brain. Following the discovery of cholinergically induced theta rhythm recorded in slice preparations of HPC,
central mechanisms underlying theta generation have been successfully studied in the in vitro conditions. In our previous work we have
attempted to record an EEG activity of three types of transected
hippocampal slices (CA1 trans-slice, CA3c trans-slice, and DG
trans-slice) examining activity of separated intrahippocampal theta generators. Data obtained in those studies revealed that dentate
gyrus isolated from generators located in CA1 and CA3c fields respectively, is not able to generate theta rhythm in the in vitro conditions. The purpose of the present study was to extend our previous
in vitro studies concerning the activity patterns of theta related cells.
This time theta cells were recorded in transacted hippocampal slices
i.e. CA1 trans-slice, CA3c trans-slice, and DG trans-slice. Sixty
cells were isolated and recorded from transected slices, including
thirty cells from CA3c (20 theta-on cells, 6 theta-off cells, and 4
gating cells), and thirty cells from CA1 (15 theta-on cells, 13 thetaoff cells, and 2 gating cells). None of DG transected slices tested
generated any synchronous field potentials and accompanying cell
activity. Supported by MNiSW grant No. N N 303 091 834.
TVII.30
THETA GENERATORS IN COMBINED TRANSECTED
HIPPOCAMPAL FORMATION SLICES
Golebiewski H., Kowalczyk T., Bocian R., Posluszny A.,
Konopacki J.
TVII.32
EFFECT OF (R)-(+)-8-OH-DPAT ON THE PHASE SHIFT
OF LOCOMOTOR ACTIVITY RHYTHM INDUCED BY
DARK PULSES IN CONSTANT LIGHT IN MICE
Bartoszewicz R., Barbacka-Surowiak G.
Rhythmic slow activity (RSA, theta) is commonly associated with the
hippocampal formation (HPC). It is generally agreed that HPC theta
rhythm is dependent on an intact septal area that act as a pacemaker
for the RSA. However, hippocampal neurons cannot be considered as
passive followers of rhythmic septal inputs. The local hippocampal
networks may also contribute to the RSA – application of the cholin-
The evidence from a variety of studies suggests the involvement
of serotonin (5-HT) in both the response of the suprachiasmatic
nuclei neurons to light and the phase-resetting properties of nonphotic stimuli. Although it is well known that 5-HT agonist causes
a phase shift of locomotor activity rhythm, there are few reports of
experiments on the influence of serotonin upon the locomotor ac-
tivity rhythm of nocturnal rodents kept under constant light (LL)
conditions. In our previous experiments we characterized the phase
response curve (PRC) for (R)-(+)-8-OH-DPAT in LL in mice and
we discovered that the shape of this PRC is similar to the PRC for
dark pulses under LL. It suggests that mechanism response for phase
shifts of locomotor activity rhythm induced by 5-HT1A agonist and
by dark pulses may be the same. Therefore, we have examined the
effect of (R)-(+)-8-OH-DPAT on the phase shifts of locomotor activity rhythm induced by dark pulses. Eight week old C57BL/10 male
mice were kept in a soundproof room in individual cages with free
access to a running wheel. The wheel-running activity rhythm was
recorded under 12:12 LD and under LL. In the LL (R)-(+)-8-OHDPAT was i.p injected every two hours during twenty-four hours.
Injections were given at 30 minutes before 4 hours dark pulses. The
analysis of the phase shifts of the locomotor activity rhythm indicated that at some time points (R)-(+)-8-OH-DPAT increases dark pulses phase-shifts whereas in other points decreases were observed.
TVII.33
CHANGES OF THE FIRING PATTERNS OF
SEROTONERGIC NEURONS IN THE RAT MEDIAN
RAPHE NUCLEUS DURING LONG LASTING
RECORDINGS
Werhun K.1, Raison S.2, Pevet P.2, Lewandowski M.1
1
University, Kraków, Poland; 2 Department of Neurobiology of
Rhythm – INCI, University of Strasbourg, Strasbourg, France
The median raphe nucleus (MRN) is one of the main sources of serotonergic ascending projections. Previous electrophysiological studies have shown that serotonergic neurons spontaneously discharge
single broad action potentials in a slow clocklike firing pattern. Subsequent studies have revealed another subpopulation of serotonin
(5-HT) containing cells, which possess the same electrophysiological attributes of classical 5-HT neurons, simultaneously displaying
bursting pattern of activity. More recently it has been shown additional class of 5-HT neurons, with relatively fast firing activity
(more than 8 Hz), implying larger diversity of 5-HT cells populations. The present data confirm formerly proposed supposition that
individual 5-HT cells are able to switch between different activity
pattern, earlier described as distinctive subpopulation of these cells.
In the present study in vivo extracellular single unit recordings were
made from the presumptively identified 5-HT neurons within the
MRN of the urethane anesthetized rat. The activity patterns were assessed by constructing interspike interval histograms. In the course
of long lasting (up to 10 hours) recordings we have found, that in
some cases individual 5-HT neurons have changed the pattern of
activity from regular to less regular, bursting or nearly silent. This
heterogeneity of firing modes is more suitable to an explanation of
the role that serotonergic system plays in diverse physiological and
pathological processes.
TVII.34
LOCALIZATION OF CRYPTOCHROMEIMMUNOPOSITIVE NEURONS IN THE VISUAL
SYSTEM OF DROSOPHILA MELANOGASTER
Damulewicz M.
Department of Cytology and Histology, Jagiellonian University,
Kraków, Poland
Cryptochrome (CRY) is a blue light absorbing protein involved
in the photic entrainment of circadian clock in the fruit fly. It
has also been suggested that CRY may play role in molecular
circadian clock that generates circadian rhythms in the visual
system. The aim of our study was to examine localization of
CRY-immunopositive neurons in the visual system of Drosophila and changes in abundance of CRY protein in these neurons. We used Drosophila transgenic lines which express green
fluorescent protein (GFP) under control of the cry promotor.
Analyses of GFP fluorescence showed that CRY is present in the
dorsal neurons DN3, and in the dorsal (LNd) and ventral lateral
neurons (LNv) and its density decreases during the day and increases during the night. In the LNv and in the processes of DN3,
the CRY abundance was the highest at ZT1 (1 h after lights-on)
and the lowest at ZT13 (1 h after lights-off) in both males and
females. In addition we found that the LNvs project to the second visual neuropil (medulla) and form CRY-immunopositive
network of many varicose processes in the medulla. We also detected numerous CRY-immunopositive processes in the fi rst optic neuropil (lamina) which origin from a single fibre extending
from the LNvs. In the lamina it divides into many thin branches
next to the retina. Our results showed for the fi rst time that the
lamina is invaded directly by processes of clock neurons which
seem to control circadian plasticity of neurons and synapses in
the lamina.
TVII.35
THE INFLUENCE OF OREXIN B ON THE ACTIVITY OF
THE INTERGENICULATE LEAFLET NEURONS
Pekala D., Blasiak T., Zabicka E., Pawlowska K., Pietrajtis K.,
Lewandowski M.
The most known function of the intergeniculate leaflet (IGL)
is regulation of circadian rhythms by integration of photic and
non-photic cues and conveying this signal to the site of main
oscillator – suprachiasmatic nuclei. Information about nonphotic cues enter the IGL from various brain nuclei, including those involved in regulation of sleep/wake states. Hypothalamic peptides – orexins has been implicated in numerous
physiological functions including maintenance of arousal and
wakefulness. Since it has been shown that fibers containing
orexins are distributed in the area of the IGL, the orexinergic
system may constitute one of the possible candidate to transmit
non-photic/arousal related cues to the IGL. Using the immunohistochemical and electrophysiological recording techniques
we evaluated the innervation of the two major types of the IGL
neurons by fibers containing orexin B (OXB) and its influence
on the activity of the IGL cells. Experiments were performed
on brain slices, obtained from adult male Wistar rats, using extracellular single unit recordings. For each individual neuron,
after reaching the stable baseline activity, the OXB (1 μM) was
applied by local pressure injection. Administered drugs evoke
an increase in the fi ring rate of the IGL neurons. Obtained data
are in agreement with the results of our immunohistochemical
staining revealing the presence of orexin fibers in the area of
the IGL.
366
TVIII: Cognition and Behavior
TVIII.01
THE COGNITIVE NEUROSCIENCE OF AUDITORY
DISTRACTION
Campbell T.
Helsinki Collegium for Advanced Studies, University of Helsinki,
Helsinki, Finland
Background sound can disrupt cognitive performance, even when
the person performing that task tries to ignore that sound and even
when the sound is unrelated to the task being performed. There are
several forms of auditory distraction, each of which may implicate
multiple cognitive processes that, in turn, could be related to multiple brain processes. One form of auditory distraction can disrupt
the performance upon a memory task that involves holding lists of
to-be-remembered digits in memory briefly, before attempting to report back those to-be-remembered items in the original order. This
auditory distraction effect occurs during a retention interval when
those items are held in memory and when the sounds are presented.
This disruption of immediate memory by auditory distraction could
be related to the generation of brain processes in response to the
presented sounds, as may be indexed by an auditory ERP protocol.
Support for the N1 hypothesis that distraction can involve factors
related to the generation of the N1 component of the auditory ERP
is offered by the finding that increases in token set size produce
a disruption of performance alongside an increase in N1. This increase in the disruption of immediate memory by auditory distraction is not associated with MMN, but rather is associated with a spatiotemporally and functionally-distinct increase in N1, which has
been shown to occur without the concomitant elicitation of P3a.
TVIII.02
DOES THE SPATIAL-NUMERICAL ASSOCIATION
INTERFERE WITH ORIENTING AND EXECUTIVE
ATTENTIONAL PROCESSING?
Gut M., Wasilewska M., Szumska I., Jaskowski P.
Department of Cognitive Psychology, University of Finance
and Management, Warszawa, Poland
Brain representations of numbers are spatially organized on the socalled mental number line (MNL). We investigated the relationship
between this spatial-numerical association and attention processes.
In the first task subjects responded to four-digit numbers (targets)
preceded by a centrally presented digit. They had to indicate the
side of occurrence of this previous digit in the target (left/right).
The digit location in the target could be congruent with its position
on MNL (e.g. “8” on the right) or incongruent (e.g. “9” on the left).
In the second task subjects assessed the parity of the central digit in
the five-digit numbers (right key = even; left key = odd). The condition was defined as congruent when the reaction side corresponded
to the digit position on MNL and incongruent when there was no
such correspondence. The results showed more accurate and faster
reactions in congruent than incongruent conditions. We conclude
that: (1) the digit magnitude representation shifts attention to the
direction of its representation on MNL, what confirms the interaction between orienting attention processes and digit positions on
MNL; (2) the incongruence between the correct response side (left/
right) and the digit spatial location on MNL induce the conflict,
which proves the involving of the executive attention system.
TVIII.03
IS PICTURE RECOGNITION MEMORY ENHANCED
THROUGH SENSORY OR COGNITIVE FACILITATION?
Nakic S.1, Pavela I.2
1
Department of Obstetrics and Gynecology, University Hospital
Sestre Milosrdnice, Zagreb, Croatia; 2 Department of Psychology,
University of Zadar, Zadar, Croatia
Different features of pictures can be used as recognition clues, and
colour is one of them. However, it is unclear whether enhanced
recognition memory by colours is due to the distinctiveness of features highlighted by colours (sensory facilitation), or it is due to the
colour representation in memory (cognitive facilitation). One way
to define mechanisms through which colour enhances memory is
to investigate the role of colour diagnosticity in picture recognition
memory which can be defined as the degree to which an object was
associated with a specific colour. Colour diagnosticity was manipulated by using pictures in two colours modes: naturally (high
colour diagnosticity) and unnaturally (low colour diagnosticity)
coloured pictures. Unnatural coloured mode was complementary
to the natural. Since there were two different versions of picture in
the encoding phase and two in the recognition phase, there were
four possible combinations of encoding and recognizing pictures.
60 participants were exposed to different combinations of encoding and recognizing pictures. Accuracy and recognition time were
measured. Results showed strong encoding-specificity effect, with
no differences in correct recognitions of naturally and unnaturally
coloured pictures. In other words, colour diagnosticity effect was
not found. Furthermore, it was shown that colour diagnosticity
takes part only during recognition time, but does not affect the
accuracy itself.
TVIII.04
PSYCHOPHYSIOLOGICAL MECHANISMS OF THE
TEENAGE AGGRESSION’S AND ITS CORRECTION
Stepanyan L., Stepanyan A., Grigoryan V.
Current research is aimed at reveal psychophysiological basis of
teenage aggression and finding ways of reducing aggressive behavior that is most essential and topical problems of modern society. To understand the investigated phenomenon it was applied
a complex approach. The Bass-Darky and Eysenk qiestionnaires,
drawing a non existing animal and Wagner’s Hand tests were used
for determining the level of aggression. Then, the influence of playing aggressive computer games was investigated. The brain visual
evoked potentials were recorded in frontal, orbito-frontal, temporal and anterior inferotemporal area before and after playing the
game. The obtained results have shown that the activity of frontal
cortex of highly aggressive subjects of both genders increased as a
result of playing the game, but in the anterior inferotemporal area
it decreased which correlated with decreased level of situational
agression. The reversed changes were observed at examinees with
a low level of aggression of both genders. Therefore, the direction
of changes depended on the initial level of aggression, but intensity of these changes depended on genderss. The correction methods
such as “Danish boxing”, “push”, “internal shout”, “pressing” and
“arttherapy” were used for psychocorrection residual aggression.
The above-mentioned correction methods led to decrease of situational aggression at high-aggressive boys.
TVIII.05
GENDER-DEPENDED ROLE OF FRONTAL AND
ORBITO-FRONTAL CORTEX AREAS IN REGULATION
OF TEENAGE CONFLICTNESS
Stepanyan A., Stepanyan L., Grigoryan V.
TVIII.07
CAN WE PREDICT THE CREATION OF FALSE
RECOGNITIONS FROM EVENT-RELATED BRAIN
POTENTIALS RECORDED DURING ENCODING?
Strozak P.
Department of Experimental Psychology, The John Paul II Catholic
The most of conflictogenic factors should be concerned as open or
latent aggression. Thus, “aggressive” computer games can be used
as an experimental model for study brain mechanisms of conflictness. The aim of this study was investigation of gender-depended
role of anterior cortex areas in regulation of teenage conflictness.
According to Utkin’s conflictness questionnaire test results subjects
were divided into 4 groups: boys and girls with a high or low level of
potential conflictness. The influence of aggressive computer gameplay was investigated. The brain visual evoked potentials were recorded from frontal and orbito-frontal cortex before and after playing
the computer game. The analysis of obtained data has shown that, as a
result of gameplay, the boys’ brain anterior cortex activity increased,
which correlated with decreasing level of conflictness. In the group
of girls the activity of anterior cortex areas was decreased. However,
there was a difference between frontal and orbito-frontal areas activity depended on the initial conflictness level. Initially high level
of orbito-frontal activity was recorded at girls with high conflictness.
The aggressive computer gameplay reduced orbito-frontal activity
that caused decrease of conflictness level. Non-conflicting girls had
initially high frontal activity which decreased through aggressive
gameplay. It is connected with weakening of cortex control and corresponding increase of conflictness level.
The poster depicts the project of research on event-related brain potentials (ERPs) recorded during the creation of false recognitions
of words. The magnitude of literature on this field focuses on the
stage of recognition when subjects decide whether or not given
words were presented to them previously. Only two recent studies
analyzed ERPs recorded during encoding when subjects mere try
to remember visually presented words which subsequently lead to
falsely recognize (or correctly reject) the corresponding associates.
However, the results of these studies are inconsistent. Urbach et al.
(2005) reported higher amplitude of LPC component (500–800 ms
poststimulus) for words not eliciting false recognitions while Geng
et al. (2007) reported higher amplitude of LPC component (500–640
ms poststimulus) for words eliciting false recognitions. The authors
claim that these effects reflect item-specific information encoding
or associative encoding, respectively. However, it seems highly improbable that the same ERP component reflects different cognitive
processes. Therefore, the current project proposes framework for
understanding that discrepancy, namely the Activation Monitoring
Theory developed by Roediger et al. (2001). Moreover, the project
points two factors that can influence the creation of false recognitions and their electrophysiological correlates during encoding: the
associative strength of words and their presentation duration.
TVIII.06
IMAGINED AND PERCEIVED ITEMS: AN EVENTRELATED POTENTIAL STUDY OF SOURCE-MEMORY
Komorowska M., Tacikowski P., Nowicka A.
TVIII.08
DYNAMICAL PROPERTIES OF “INTERNAL CLOCK”
AS A NEURAL BASIS OF INDIVIDUAL DIFFERENCES
IN THE LEVEL OF INTELLIGENCE
Dreszer J.1, Szelag E.2, Osinski G.3
1
Faculty of Fine Arts, Nicolaus Copernicus University, Toruń,
Poland; 2 Laboratory of Neuropsychology, Nencki Institute of
Experimental Biology PAS, Warszawa, Poland; 3 Department of
Informatics, Nicolaus Copernicus University, Toruń, Poland
Mental processes that are initiated without any input from outside
world may differ from those related to the direct external stimulation. Specifically, a question arises whether memory traces for
previously imagined items may differ from memory traces for
perceived items. The aim of our study was to test this issue, using the event-related potentials (ERP). It is well documented that
remembered old items elicit more positive-going ERPs than correctly judged new items. This so-called “old/new effect” indexes
neural activity associated with correct retrieval of information
about a prior event. In this study the old/new effect was investigated separately for previously imagined and perceived items.
In the first part of the study, word labels of common objects were
presented. Half of them were followed by a colour picture of the
corresponding object. The other half was followed by a black
screen, signalling to the participants to mentally visualize the object. In the second part, the participants discriminated between
new words, words corresponding to previously perceived or imagined pictures. Correctly identified old items were associated with
more activity than correctly judged new items over the left parietal areas. Importantly, this statistically significant old/new effect,
representing the recollection index, was greater for imagined than
for perceived items. Thus brain representations of imagined items
might be more detailed and precise than those of real objects.
Neuropsychological evidence has suggested that both intelligence and
temporal resolution in information processing may be determined by
similar neural mechanisms, related to internal timing mechanism. Personal tapping tempo (PTT) can be considered as a promising measure
of such “internal clock” properties, as it reflects spontaneous tempo in
human motor activity. As the existing studies do not provide detailed
description of temporal characteristics of PTT, conclusions on temporal control underling timing and intelligence are still not clear. The
present study investigated in 55 highly intelligent and 50 average intelligent students dynamical properties of temporal control of repetitive
finger movements in PTT. Nonlinear elements for reconstruction of
dynamical properties of PTT were performed. We found fractal properties of tapping performance. Additionally, advanced mathematical
analyzes showed grouping in frequencies of PTT, which constituted
characteristic optima of subjects’ performance. In these optima intelligence-related differences were revealed. These findings supported the
notion that ’internal clock system’ in the brain could be fractal in its
nature which has a different structure in highly and average intelligent
individuals. The study was supported by Grant N N106 109636 from
Ministry of National Education and Science.
368
TVIII.09
NEUROANATOMICAL CORRELATES OF FAST
FORWORD TRAINING: AN fMRI STUDY
Lewandowska M.1, Piatkowska-Janko E.2, Bogorodzki P.2,
Wolak T.3, Szelag E.1
1
Biology PAS, Warszawa, Poland; 2 Institute of Radioelectronics,
Warsaw University of Technology, Warszawa, Poland; 3 Institute of
Physiology and Pathology of Hearing, Warszawa, Poland
than in women in dorsolateral prefrontal (BA 9, 46), parietal (BA
7, 40), and occipital (18, 19) cortices. In contrast, women activated
insular (BA 13), temporal (BA 21, 22) and ventrolateral prefrontal (BA 44) cortices. Differences between men and women in the
patterns of brain activity might reflect distinct strategies used in
the weather prediction probabilistic classification task. Our results
indicate that neural activity underlying implicit memory differs
between sexes, despite of lack of differences in behavioral efficiency.
Using fMRI we tested brain activations involved in temporal
processing following Fast ForWord (FFW) training in 11 young
volunteers. They performed the Temporal Order Judgment task in
two sessions: before and after the training. The task was to report
the order of two sounds presented in rapid succession. Each pair
consisted of a short (10-ms) and a long (50-ms) sound separated by
Inter-Stimulus-Intervals of 160 ms (easy condition), 60 ms (medium) and 10 ms (difficult). FFW training was applied for ca. 8
weeks with four 1-hour sessions per week. The brain activations
during particular conditions (“before vs. after” and “after vs. before”) the training were compared using paired sample t-tests from
SPM5. ‘Before vs. after’ comparisons resulted in the following activations: (1) easy condition – left cerebellum, (2) medium condition – cerebellum and insula bilaterally, left thalamus and putamen
and moreover, bilaterally selected areas of the temporal, frontal
and parietal lobes, (3) difficult condition – left insula and the left
temporal, frontal and parietal gyri. “After vs. before” comparisons
showed no significant differences for each condition, thus, indicating no increase of activation “after” than “before” the training.
These results support the involvement of above structures in timing and suggest decreased brain activation in timing tasks following FFW training. We thank Scientific Learning for the opportunity to use FFW grant MSHE no.: 1082/P01/2006/31.
TVIII.11
INTERHEMISPHERIC RELATIONS IN ATTENTION
EVIDENCED BY THE LATERALIZED ATTENTION
NETWORK TEST (LANT)
Asanowicz D., Wolski P.
Institute of Psychology, Jagiellonian University, Kraków, Poland
TVIII.10
SEX DIFFERENCES IN IMPLICIT MEMORY:
AN fMRI STUDY USING THE WEATHER
PREDICTION PROBABILISTIC CLASSIFICATION TASK
Pilacinski A.1, Wolak T.2, Grabowska A.3, Krolicki L.4,
Szatkowska I. 3
1
Department of Cognitive Neurology, Hertie Institute for Clinical
Brain Research, Tuebingen, Germany; 2 Bioimaging Research Center,
Institute of Physiology and Pathology of Hearing, Warszawa, Poland;
3
Biology PAS, Warszawa, Poland; 4 Department of Nuclear Medicine,
Warsaw Medical University, Warszawa, Poland
Several recent studies have shown sex-related differences in the
neural organization of memory processes. However, as the majority of investigations involved explicit memory tasks, little is known
about potential differences between men and women in the organization of implicit memory processes. Our study addressed this
issue. We used functional magnetic resonance imaging (fMRI) to
compare the patterns of brain activity in men and women performing the weather prediction probabilistic classification task which is
a tool for assessing implicit learning. In this task, subjects learn to
predict the weather using associations that are formed gradually
across many trials, because of the probabilistic nature of the cueoutcome relationships. Although both men and women exhibited
similar level of behavioral performance, between-sex group analysis of the BOLD response demonstrated greater activity in men
Attention has been convincingly demonstrated to involve anatomically and functionally distinct networks that subserve conflict resolution, spatial orienting and alerting. On the one hand,
partially independent symmetric attention networks can be postulated within the two brain hemispheres (Zaidel 1995). On the other
hand, large body of data, mostly clinical, suggests asymmetric
control of attention (Heilman and Van Den Abell 1980). Not much
is known at present on the nature of interhemispheric relations in
attention. To address this issue, the Lateralized Attention Network
Test (LANT) was designed. The introductory study of Greene et
al. (2007) has suggested some hemispheric independence and similarity but no clear asymmetry. To gain better insight we conducted three consequent experiments recruiting 170 participants. We
supplemented the original LANT procedure with modifications
that allowed comparisons between goal-directed and stimulusdriven control of attention. The results show that interhemispheric
relations within the attentional networks do depend on whether the
“endogenous” or the “exogenous” control dominates in the task.
TVIII.12
ANXIETY-RELATED BEHAVIOR IN RATS WITH
CONSTITUTIONALLY ALTERED SEROTONERGIC
ACTIVITY
Mokrovic G., Cicin-Sain L.
Department of Molecular Biology, Rudjer Boskovic Institute, Zagreb,
Croatia
Serotonin (5HT, 5-hydroxytryptamine) is involved in regulation
of both central and peripheral physiological functions, and disturbances in serotonergic neurotransmission are common in neuropsychiatric disorders. Using selective breeding, we have developed
Wistar-Zagreb 5HT rats that include two sublines: one with high
(high-5HT) and other with low (low-5HT) levels of peripheral
(platelet) serotonin and activity of 5HT transporter, representing a
rodent model with constitutionally altered serotonin homeostasis.
We hypothesized that in these animals, beside peripheral, brain
serotonergic activity is also altered which should have effect on
their behavioral response. In the present study, we have tested the
anxiety-related behavior of 5HT sublines (3 months old males,
n=20–21 per subline) in elevated plus maze and open field paradigms. Animals from high-5HT subline had lower score in time
spent in open arms and number of entries to the open arms of
elevated plus maze in comparison to low-5HT subline (P<0.05).
Also, the score of head dip time in open arms was lower in high-
5HT subline (P<0.005). In open field test, high-5HT subline had
lower score in total rearing time (P<0.005). Altogether, these results confirm results of previous behavioral tests (hole-board and
zero maze) showing differences in anxiety-related behavior in
Wistar-Zagreb 5HT rats and indicating higher level of anxietyrelated behavior in high-5HT subline.
TVIII.13
COLOCALISATION OF GLUCOCORTICOID AND 5-HT1A
RECEPTOR IMMUNOREACTIVITY-EXPRESSING
CELLS IN THE BRAIN STRUCTURES OF LOW AND
HIGH ANXIETY RATS
Lehner M.1, Taracha E.1, Maciejak P.1, Szyndler J.2,
Skorzewska A.1, Turzynska D.1, Sobolewska A.1,
Wislowska-Stanek A.2, Hamed A.2, Bidzinski A.1, Plaznik A.1
1
Department of Neurochemistry, Instituite of Psychiatry and
Clinical Pharmacology, Medical University, Warszawa, Poland
In recent years we have elaborated an animal model to examine the neurochemical background of differences in the individual responses to conditioned aversive stimuli, using the
strength of a rat contextual fear test, as a discriminating variable: low responders (LR), i.e. rats with duration of a freezing
response one standard error, or more, below the mean value
and high responders (HR), i.e. rats with duration of a freezing
response one standard error, or more, above the mean value.
It was found that 1.5 h after a testing session of contextual
fear test, the LR animals showed a higher density of 5-HT1A
and glucocorticoid immunoreactivity-expressing cells (GRsir) in the cortical M2 area and hippocampal dentate gyrus as
well as an increased number co-expressing 5-HT1A /GRs-ir in
the same areas. The HR rats had a significantly higher concentration of 5-HT1A and GRs-ir in the basolateral amygdala.
The present data add more arguments for the neurobiological
background of differences in individual responses to aversive
conditioned stimuli.
TVIII.14
SOCIAL MODULATION OF AVERSIVE LEARNING
IN RATS
Knapska E.1, Mikosz M.1, Sadowska J.1, Maren S.2, Werka T.1
1
Biology PAS, Warszawa, Poland; 2 Department of Psychology,
University of Michigan, Ann Arbor, MI, USA
It is well known that emotions participate in regulation of social
behaviors and that the emotional states displayed by a conspecific can seriously affect the behavior of other animals. In its
simplest forms empathy can be characterized as the capacity to
be affected by and/or share the emotional state of another. However, to date, relatively little is known about the mechanisms by
which the animals that are not in a direct danger can share emotions. In the present study we used the model of between-subject transfer of fear to characterize the social interaction during
which fear is transmitted, as well as the effects of socially transmitted fear on behavior of its recipients. We found that: (1) during social interaction with a recently fear conditioned partner,
observers and demonstrators exhibit social exploratory behaviors rather than aggressive behaviors; (2) learning in a shockmotivated shuttle avoidance task is facilitated in rats that under-
went the social interaction with a partner that had been either
fear conditioned or trained in two-way avoidance; (3) a brief
social interaction with a recently fear conditioned partner immediately before fear conditioning improves conditioned freezing measured on the next day; this effect can be also seen in rats
that are unfamiliar to each other. Collectively, the obtained data
suggest that a brief social interaction with a cage-mate that had
undergone an aversive learning experience promotes aversive
learning in an otherwise naïve animal.
TVIII.15
DIFFERENTIAL CONTRIBUTION OF DORSAL
HIPPOCAMPUS IN THE REORGANIZATION OF
HOMOGENOUS ALLOTHETIC OR IDIOTHETIC
SPATIAL REPRESENTATION IN RATS
Adamska I., Popowski P., Wesierska M.
The hippocampus is engaged in the processing of visual (allothetic) and self-motion (idiothetic) information for spatial
memory function. Beyond memory, it is involved in cognitive coordination processes (Wesierska at al. 2005). We hypothesised that the hippocampus is also critical for the reorganization of spatial representations. To study this we used a
place avoidance (PA) method to create homogenous allothetic
(Room: R+) or idiothetic (Arena: A+) spatial representations.
We then rearranged these representations according to the A+
or the R+. In the PA task hippocampal lesioned (HL) and intact
(I) rats learn to avoid a place on the arena where shocks are administered. In the R+ variant this place was in a fi xed position
according to the room frame (lit room, rotating arena, waterdissolved proximal cues); the A+ variant was represented by a
fi xed position according to the arena information (dark, rotating arena). The HL rats were impaired during acquisition of
avoidance in the R+ (F1, 14=13.97; P<0.002) but not in the A+
variant. When spatial conditions were rearranged all lesioned
rats showed impaired learning (from R+ to A+: F1, 13=12.04;
P<0.004; from A+ to R+: F1, 14=10.59; P<0.005). Our results
suggest that acquisition of allothetic representation, compared
to idiothetic representation is more highly hippocampal-dependent. Conversely, an unimpaired hippocampus seems to be
necessary for the reorganisation of spatial representation. Supported by grant 3120/B/P01/2007/33.
TVIII.16
RECIPROCAL PATTERNS OF c-Fos EXPRESSION
IN THE MEDIAL PREFRONTAL CORTEX AND
AMYGDALA AFTER EXTINCTION AND RENEWAL
OF CONDITIONED FEAR IN RATS
Knapska E.1, Maren S.2
1
Biology PAS, Warszawa, Poland; 2 Department of Psychology,
University of Michigan, Ann Arbor, MI, USA
Substantial evidence indicates that extinguished fear can be
recovered after a change in experimental context (i.e., the renewal effect). The aim of this study was to characterize the
neural circuitry underlying the retrieval of extinguished fear
370
memories using c-Fos immunohistochemistry. Firstly, rats
received auditory fear conditioning. Subsequently, they were
extinguished by presenting CS-alone trials in either the same
context as conditioning or in a second context. Then, all rats
were tested for their fear of the auditory CS in the second context and sacrificed 90 min after testing. The presentation of
the extinguished CS outside of the extinction context resulted
in renewal of the freezing response relative to animals tested
to the CS in the extinction context. In addition, the renewal of
fear was associated with c-Fos expression in the prelimbic division of the medial prefrontal cortex, the lateral and basolateral
nuclei of the amygdala, and the medial division of the central
nucleus of the amygdala. In contrast, the presentation of the
CS in the extinction context induced c-Fos expression in the
infralimbic cortex, the intercalated nuclei of the amygdala and
the dentate gyrus. Hippocampal areas CA1 and CA3 exhibited
c-Fos expression when the CS was presented in either context.
These data suggest that the context-specificity of extinction
may be mediated by prefrontal modulation of amygdala activity, and that the hippocampus may have a fundamental role in
contextual memory retrieval.
TVIII.17
LOCAL TRANSLATION – THE MECHANISM
OF LONG-TERM MEMORY FORMATION
IN THE ABSENCE OF ALPHA CaMKII
AUTOPHOSPHORYLATION
Radwanska K.1, Irvine E.2, Schenatto-Pereira G.3,
Dutra-Moraes M.3, Giese K.4
1
Department of Molecular and Cellular Neuroscience, Nencki
Institute of Experimental Biology PAS, Warszawa, Poland; 2 Wolfson
Institute for Biomedical Research, UCL, London, UK; 3 Neuroscience
Centre, Federal University of Minas Gerais, Belo Horizonte, Brazil;
4
Institute of Psychiatry, Kings’ College London, London, UK
Alpha calcium and calmoduline-dependent kinase II (CaMKII)
is the major protein in glutamateric neurons in the forebrain.
Its activity is regulated by autophosphorylation on the threonine
286. CaMKII-T286A mutant mice have severe deficits in context
memory formation. They form fear memory of the context in
fear conditioning task only after very intensive training with 5
shocks. Here we present the data showing that foreground fear
conditioning training does not induce expression of any of the
analysed immediate early genes (c-Fos, Zif268, Nur77 or JunB)
in CaMKII-T286A mutant mice. Furthermore, long-term fear
memory in the mutants cannot be blocked by administration of
mRNA or protein synthesis inhibitors (actinomycin D or anisomycin) into dorsal hippocampus. Moreover, as revealed by Illumina microarrays, fear memory training does not induce context-shock association specific expression in the hippocampus.
On the other hand, the training of the CaMKII-T286A mutant
mice induces in the hippocampus expression of locally-translated proteins, Arc and PSD-95. In addition, both training-induced
PSD-95 expression and long-term fear memory can be blocked
by intrahippocampal administration of rapamycin (inhibitor of
mammalian target of rapamycin kinase, the main regulator of
local translation). Thus, our data strengthen the notion that local
translation of PSD-95 in the dorsal hippocampus is the mechanism of long-term memory formation in the absence of alpha
CaMKII autophosphorylation.
TVIII.18
IMMINENT VS. REMOTE DANGER: EMOTIONAL
REACTIONS AND PATTERNS OF c-Fos EXPRESSION
IN THE RAT BRAIN
Mikosz M.1, Knapska E.1, Nikolaew J.2, Solarska U.1, Serzysko K.1,
Sadowska J.1, Werka T.1
1
It has been shown that emotional states displayed by animals are
able to influence their conspecifics’ behavior and that such transferred emotions help animals to adapt to specific requirements
of changing environment. Animals that are not in direct danger
can share fear by either observation of a conspecific in danger or
being informed by a conspecific about danger. To date, relatively
little is known about mechanisms by which the emotional transfer
occurs. Aiming to gain more insights into the neural substrates
of two types of socially transferred fear, we designed behavioral
models of indirect emotional stimulation. To allow observation of
a conspecific in danger, we placed a rat designated as an observer
in a safe compartment of two-compartment conditioning box and
exposed the animal to a demonstrator being fear-conditioned in
the other compartment. To model a situation in which an observer
cannot witness a demonstrator in direct danger, we used a rat that
had previously undergone fear conditioning as stimulus source
for the other individual. Witnessing conspecific’s distress evoked
an increase in defensive responses and a decrease in exploratory
behaviors. On the other hand, interaction with a stressed partner
increased behaviors directed towards gaining information (sniffing and allogrooming). Activation of various brain structures
following observation of a conspecific in danger and interaction
with a conspecific that underwent a stressful stimulation were
analyzed.
TVIII.19
IL-6 DEFICIENCY ALTERS COGNITIVE PROCESSES
IN MICE
Winnicka M.1, Bialuk I.1, Hryniewicz A.1, Mencel J.1,
Kaminski K.2
1
Department of General and Experimental Pathology, 2 Department
of Cardiology, Medical University of Bialystok, Białystok, Poland
Interleukin-6 (IL-6) in addition to its role in the immune system
has the potential to modulate several brain functions including
learning and memory processes. In the present study we investigated the role of IL-6 in CNS function in male mice not expressing IL-6 (C57BL/6J IL6-/-tm 1 Kopf ) and wild type mice (WT)
used as controls. The animals were kept in standard conditions
with water and food available ad libitum except during experiments. All testing took place between 8.30 AM and 12.30 PM. Each
group consisted of 13 animals. In order to evaluate a role of endogenous IL-6 in cognitive functions “object recognition test” for
the evaluation of recognition memory was used. In an attempt to
evaluate whether observed effect was memory specific, the level
of anxiety and psychomotor activity of mice was evaluated in an
“elevated plus maze” test and in an open field, respectively. Recognition memory, measured by the difference in exploration of
the new object and a duplicate of the familiar one, presented 1 h
earlier, was impaired in IL-6 deficient mice. Moreover, lack of
IL-6 significantly attenuated locomotor and exploratory activity
measured in an open field test and enhanced anxiety evaluated in
an “elevated plus maze” test. Results of this study indicate that
IL-6 deficiency impairs recognition memory, attenuates locomotor and exploratory activity and enhances anxiety in mice. The
study was supported by the Polish Ministry of Science grant Nr
2P05B01826.
TVIII.20
NEW HIPPOCAMPAL NEURONS ARE NOT
OBLIGATORY FOR MEMORY FORMATION;
MICE WITH NO ADULT BRAIN NEUROGENESIS
SHOW LEARNING
Jaholkowski P.1, Kiryk A.1, Jedynak P.1, Ben Abdallah N.2,
Knapska E.1, Kowalczyk A.1, Piechal A.3, Blecharz-Klin K.3,
Widy-Tyszkiewicz E.3, Wilczynski G.1, Lipp H.2, Kaczmarek L.1,
Filipkowski R.1
1
2
Department of Neuroanatomy and Behaviour, Anatomy Institute,
University of Zurich, Zurich, Switzerland; 3 Department of
Experimental and Clinical Physiology, Medical University
of Warsaw, Warszawa, Poland
New neurons are produced in the brains of adult animals, including humans, throughout the lifespan. Since one of the site of adult
brain neurogenesis is the hippocampal formation, a brain structure
involved in learning and memory, new neurons were expected to
be involved in these phenomena. However, the very evidence supporting this hypothesis remains limited, inconsistent and in most
cases indirect. Also, the experiments in which adult neurogenesis
is blocked use irradiation and drugs known for their side effects.
We used a novel approach, cyclin D2 knock-out mice (D2 KO
mice), specifically lacking adult brain neurogenesis (which was
verified using DCX and NeuN staining) to verify its importance
in learning and memory. D2 KO mice and their wild type siblings were tested in several behavioral paradigms including those
in which the role of adult neurogenesis has been postulated. D2
KO mice showed no impairment in sensorimotor tests with only
sensory impairment in an olfaction-dependent task. However, D2
KO mice showed proper procedural learning as well as learning
in context (including remote memory), cue and trace fear-conditioning, Morris water maze, novel object recognition test, and in a
multifunctional behavioral system – Intellicages. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis
has previously been strongly suggested. Support – MNiSW grant
no N303 051 31/1624.
TVIII.21
PHENOTYPING OF TRANSGENIC MICE: COMPARISON
OF INTELLICAGE–SYSTEM TO AUTOMATIC
MONITORING OF BEHAVIOR IN SOCIAL CONTEXT
WITH STANDARD TESTS
Kiryk A., Kaczmarek L.
There is a growing need for new strategies allowing for screening, monitoring and phenotyping of genetically modified ani-
mals. One of the instruments is IntelliCage system, an automated testing device allowing to investigate behavior in social
groups of mice in repetitive conditions, without excessive
contact with experimenter. We compared results of 4 strains
of genetically modified mice tested individually in standard
battery of tests with the animal performance in social context
in IntelliCage. The overall behavior of tested strains, namely
D2 knock-out mice lacked adult neurogenesis, GLT1 knock-out
mice deprived of glial glutamate transporter, APP.V717I transgenic mice expressing the mutant human amyloid precursor
protein and Dicer mice with forebrain specific and inducible inactivation of Dicer gene, were at fi rst analyzed in sensorimotor
tests, open field and elevated plus maze. Activity, exploration
and anxiety level were then investigated in IntelliCage system
and we found that all the tests were able to detect differences in
phenotype. Furthermore, learning and memory tests were conducted in IntelliCage, e.g. place preference and confronted with
traditional learning tasks, such as Morris water maze. Again,
IntelliCage as well as standard tests differentiated mice with
mutation from their wild-type siblings. We also found on example of APP.V717I transgenic mice prospects for investigation
the effect of the social interaction on learning in IntelliCage
system.
TVIII.22
OLD TRANSGENIC RATS WITH OVEREXPRESSION
OF MMP-9 ARE MORE ACTIVE THAN CONTROLS
AND REMEMBER BETTER IN BEHAVIORAL TESTS
Wawrzyniak M.1, Kiryk A.1, Lioudyno V.2, Meyza K.3,
Owczarek D.1, Kaczmarek L.1
1
2
Pavlov Department of Physiology, Institute for Experimental
Medicine RAMS, Sankt-Petersburg, Russia; 3 Department of
Neurophysiology, Nencki Institute of Experimental Biology PAS,
Warszawa, Poland
Matrix metalloproteinases are a major group of enzymes regulating cell-matrix composition, which are essential for many
biological processes. Matrix Metalloproteinase-9 (MMP-9)
has recently emerged as an important molecule in control of
extracellular proteolysis in the synaptic plasticity. Using conventional transgenesis, we have created rats with overexpression of MMP-9 limited to the neurons of the hippocampus, cerebral cortex and cerebellum (MMP-9-gene is under control of
neuronal, synapsin-1 promoter). A battery of behavioral tests,
including Open Field and Elevated Plus Maze tests, revealed
no significant difference between young transgenic and control rats. However, age-related changes in expression level
of MMP-9 in wild type rats led to some dysfunctions in behavior, as we noticed differences in general activity between
the transgenic and wild type rats increasing with age. The old
transgenic rats demonstrated higher activity and better motor
functions and coordination than controls. Moreover, the level
of anxiety was decreased in the old transgenic rats in comparison with their wild type siblings. Also, the old transgenic
rats showed better taste memory than wild type. These results
strongly suggest that MMP-9 may have an important role in
control of the behavior of animals.
372
TVIII.23
INSIGHTS INTO SPATIAL MEMORY FORMATION
IN RETROSPLENIAL CORTEX
Czajkowski R.1, Wiltgen B.1, Balaji J.1, Rogerson T.1,
Guzman-Karlsson M.1, Barth A.2, Silva A.1
1
Department of Neurobiology, UCLA, Los Angeles, CA, USA;
2
Department of Biological Sciences, Carnegie Mellon University,
Pittsburgh, PA, USA
The development of two-photon imaging techniques has created
new ways to study the function of the mouse brain. We are using this method to monitor in vivo the activation of a FosGFP reporter in the retrosplenial cortex (RSC) in mice with chronically
implanted optical windows. This study was designed to explore
the mechanisms of RSC involvement in spatial memory encoding.
RSC receives projections from the anterior thalamus and the CA1
region of hippocampus; it is also reciprocally connected with the
subiculum, anterior cingulate and parietal cortices. Therefore this
structure has been proposed to have a critical role in the circuitry
responsible for spatial learning and memory. For the imaging experiment mice were trained for 10 days alternatively in a “spatial”
or in a “cued” version of the Morris water maze task (MWM). In
the “spatial” version (days 1, 3, 5, 7 and 9 of training) mice could
use extra-maze cues in the experimental room. In the “cued” version (days 2, 4, 6, 8 and 10), the extra-maze cues were blocked
with a curtain placed around the pool and the platform position
was marked with metal rod. On days 7–10, 90 minutes after the 4th
and 5th sessions, specific regions of the RSC were imaged in lightly
anesthetized FosGFP mice. Analysis revealed that approximately
30% of cells imaged showed reliable changes in activity when alternating between the spatial and cued task. Among those cells,
the majority showed higher activity after the spatial task than after
the cued task.
TVIII.24
EMG ACTIVITY IN RESPONSE TO DYNAMIC
AND STATIC FACIAL EXPRESSIONS
Rymarczyk K.1, Biele C.1, Majczynski H.1, Gut M.2,
Jankowiak-Siuda K.3, Grabowska A.1
1
Biology PAS, Warszawa, Poland; 2 Department of Cognitive
Psychology, University of Finance and Management, Warszawa,
Poland; 3 Department of Experimental Neuropsychology, Warsaw
School of Social Sciences and Humanities, Warszawa, Poland
Facial expressions are complex signals caused by rapid changes in
facial muscular activity that are brief and last only a few seconds.
The suggestion that dynamic facial expressions of emotion induce
more evident facial mimicry than static ones remains controversial. We investigated this issue by recording EMG from the three
muscles: corrugator supercilii, zygomatic major and orbicularis
oculi. Dynamic (video movie) and static (pictures of faces with
equivalent static emotion) facial expressions of four emotions:
anger, fear, surprise and happiness were presented. The analysis
of data showed that subjects reacted spontaneously and rapidly to
happy faces with increased zygomatic and orbicularis oculi EMG
activity and with decreased corrugator EMG activity. In both zygomatic and orbicularis oculi muscles alteration of activity was
greater in response to dynamic than to static stimuli. Moreover
angry faces evoked alteration of corrugator and orbicularis oculi
EMG activity, although dynamic expressions evoked stronger re-
action in orbicularis oculi only. Unexpectedly static angry faces
evoked greater alterations in corrugator EMG activity than dynamic faces. Two other expressions of emotion: fear and surprise
did not evoke significant changes in EMG activity of none of three
muscles. The obtained results only partially confirmed the hypothesis that the dynamic stimuli evoked stronger facial muscle
reactions.
TVIII.25
THE KNOWLEDGE OF THE NEUROSCIENTIST
MISSES THE HAND BUT HITS THE MIND OF
NEUROREHABILITATION PROFESSIONAL
Kinalski R.
Department of Clinical Neurophysiology in the Physiotherapy Chair,
University of Cosmetology and Health Care in Bialystok, Białystok,
Poland
The professional of neurorehabilitation, evaluates the neuromuscular dysfunctions of man based mostly on the result of the coded
test than the instrumental ones. Such professional, in the EvidenceBased Medicine era, needs the suitable education (Kinalski 2008).
The experimental and clinical neurophysiologists should be more
often the partners of the interdisciplinary research. At present in
Poland take place two international congresses, in which clinicians, who are interested to utilize in clinical practice the results of
the preclinical research participate actively (http://ptreh.home.pl,
http://www.pns2009.pl ). The aim of the report is to show: (1) how
often the clinicians participate actively in the International Congresses of the Polish Neuroscience Society organized since 1991;
(2) what type of research results were presented by the clinicians
on the symposia and poster sessions; (3) which of the domestic and
the foreign establishments were affiliated by the clinicians.
POSTER SESSION III
H: History of Neuroscience in Poland
H.01
HOW DID THE INSTITUTE OF EXPERIMENTAL
BIOLOGY COME TO BE NAMED AFTER
MARCELI NENCKI (1847–1901)?
Leszczynska A., Majczynski H., Slawinska U.
Marceli Nencki was a great Polish scientist of the XIX century.
He began his research at the University of Berne, Switzerland, in
1872. In 1876 he was appointed Associate Professor there, and one
year later he became a full Professor and Director of the Institute of
Medical Chemistry. In 1891 Marceli Nencki accepted an invitation
to organize, together with Ivan P. Pavlov, the Institute of Experimental Medicine in St. Petersburg, where he spent the last decade
of his life. In research he concentrated on topics related to urea
synthesis, the chemistry of purines, and biological oxidation of
aromatic compounds. He also examined the structure of proteins,
enzymatic processes in the intestine, and bacterial biochemistry.
Among Nencki’s greatest successes was showing, together with
Leon Marchlewski, a close chemical relationship between hemo-
Poster Session III 373
globin and chlorophyll. The idea to establish the research institute
named after Marceli Nencki was born shortly after his death in
1901. Among his friends who pushed forward this idea, the most
effective was Nadine Sieber-Shumova, his close co-worker from
Berne and St. Petersburg. However, many years were to pass until
finally in 1918/19 the Nencki Institute was founded. Today, the
Nencki Institute, where the neurobiology, neurophysiology, biochemistry and cell biology are widely represented, is the only research centre in Poland where the investigations are performed
from the molecular to the whole animal and human subject level.
H.02
NAPOLEON NIKODEM CYBULSKI (1854–1919)
– THE FOUNDER OF ELECTROPHYSIOLOGICAL
SCHOOL IN CRACOW
Smyda J., Wrobel A.
Napoleon Cybulski was one of the most prominent Polish physiologists. He defended his PhD thesis in 1885 and became the Chair
of the Department of Physiology at the Faculty of Medicine of
Jagiellonian University, till 1919. He is known as a founder of the
physiology school in Cracow. One of his greatest achievements
was the construction of a device for precise measurements of
blood movement in the vessels – the photohemotachometer. It provided a better insight into the physiology and pathophysiology of
the circulatory system. In the field of endocrinology Cybulski, together with Szymonowicz, found out that adrenal extracts contain
biologically active substances that elevate blood pressure. He was
also a constructor of an extremely sophisticated microcalorimeter
to measure the quantity of heat produced during isolated muscle
contraction. He applied, for the first time, condensator discharges
to stimulate nerves and analyzed changes in the excitability of the
muscles. Cybulski proved that the cause of the electrical excitability of tissue depends on the electrical energy and the time of its duration. Together with Adolf Beck, he performed the experiments
on the sensory centers in the central nervous system by means of
the observations of the electrical evoked potentials. They discovered the continuous electrical oscillations in the brain and recorded the negative electrical potentials in brain areas. This invention
had the great contribution to the development of physiology.
H.03
IMPACT OF ORGANIC SEMIOLOGY ON
DEVELOPMENT OF NEUROLOGY IN THE WORK
OF JOSEPH JULES FRANCOIS FELIX BABIŃSKI
(1857–1932)
Wasowicz M., Barszcz K.
Department of Morphological Sciences, Faculty of Veterinary
Medicine, Warsaw University of Life Sciences, Warszawa, Poland
Joseph Jules François Félix Babiński was a son of emigrants from
Poland. He was born in Paris where he also went to school and
studied medicine. He graduated with honours in 1879. He made his
training as an apprentice of Cornil, Vulpian and Buequoy. When he
was assistant to Jean-Martin Charcot, he chose his specialization in
internal medicine and neurology, soon achieving mastery in these
subjects. In 1890 he was appointed head of the Pitié Hospital and
worked there till the end of his life. He wrote over 300 papers on
physiology of the nervous system and neuropathology. Babiński is
the author of organic semiology of hemiplegia and paraplegia, which
helps to differentiate them from functional and hysterical paralysis.
He discovered most of the pyramidal symptoms. In his studies on
defense reflex he defined its relationship with injuries to the pyramidal tract, named the “Babiński Sign”, a test known also as Babiński
reflex, introduced to neurology and widely used to assess upper motor neuron disease. Symptomatology of cerebellar diseases helped to
differentiate cerebellar disorders from atrial disorders, enabling to
determine such symptoms as: hypermetria, asynergy, adiadochokinesis, tremor, catalepsy. Babiński is to be remembered as a pioneer
of neurosurgery in France. He was an extremely modest person of
great intuition and of analytical and synthetic mind. He was an extraordinary researcher and clinician. He died on 29.10.1932 and was
buried in Montmorency cemetery near Paris.
H.04
ADOLF BECK (1863–1942) AND HIS SHARE
IN DEVELOPMENT OF ELECTROPHYSIOLOGY
Barszcz K., Wasowicz M.
Department of Morphological Sciences, Faculty of Veterinary
Medicine, Warsaw University of Life Sciences, Warszawa, Poland
Adolf Beck lived in years 1863–1942. He graduated from the Jagiellonian University and was an outstanding physiologist, pupil and the
closest collaborator of Prof. Napoleon Cybulski. In 1890 he became
a PhD in medical sciences. His research was devoted to the physiology of the central nervous system and opened the fundamentals of
electrophysiology and in consequence, to electroencephalography.
Cybulski’s and Beck’s studies had a pioneering character and they
were the first to discover electric activity of the brain and changes
of this activity in response to various stimuli. The first Beck paper
was published in German (1890) and produced response from English researchers who claimed the primacy of Richard Caton (1875).
However, the Caton studies differed from Beck’s experiments and
were not known to him before. Beck’s didactic and academic work
has to be underlined when presenting his profile. At 32, as Associate
Professor, he became chair of Physiology Department at Medical
Faculty of the Jan Kazimierz University in Lviv and three years later
he was promoted to Full Professor. He worked as dean of Medical
Faculty and later as chancellor. He was the author and co-author
of many manuals and original scientific papers. His book entitled
“Physiology of the Central Nervous System”, published in 1913,
meets all the requirements of a modern manual for neurophysiology.
He published a manual entitled “Human Physiology”, first in 1915
together with Napoleon Cybulski and then, in 1922, by himself.
H.05
EDWARD FLATAU (1868–1932) – THE CLASSIC
OF POLISH NEUROLOGY, CO-FOUNDER OF THE
NENCKI INSTITUTE
Oderfeld-Nowak B.1, Potkaj A.2
1
Institute of Experimental Biology PAS, Warszawa, Poland; 2 Warsaw
School of Social Sciences and Humanities, Warszawa, Poland
Edward Flatau established neurobiological and neuropathological
sciences in Poland and at the same time was an outstanding doctor.
He was born in Płock, spent several years studying and working
abroad, in Moscow and Berlin. In 1894 he wrote a key work “Atlas
of the Human Brain and the Course of the Nerve-Fibres” which was
published in many languages. He formulated the statement, known
374
as Flatau`s Law, that “greater the length of the fibres in the spinal
cord the closer they are situated to the periphery”. For this work he
received Ph.D. in medical sciences. By 1899 he returned to Poland,
as a scientist with a world known name. For many years he shared
his responsibilities as experimentalist and neurologist between the
laboratory and the hospital (he was the head of neurology in the
Jewish Hospital in Warsaw). He had a large private practice. He
is the author of more than 100 publications in many languages.
His most referenced book is classical book about Migraine (1912).
Another fundamental paper was on progressive torsion spasm in
children. He established the first neurobiological laboratory in 1911
in Warsaw which he headed until 1923. From the initiative of the
directors of Warsaw Scientific Society` laboratories: Edward Flatau (Neurobiological Laboratory), Kazimierz Białaszewicz (Physiological Laboratory) and Romuald Minkiewicz (General Biology),
an autonomous organization was formed in 1918 under the name of
Marceli Nencki Institute of Experimental Biology.
H.06
JAN PILZ (1870–1930) – THE PIONEER OF POLISH
NEUROLOGY
Magowska A.
Department of the History of Medical Sciences, University of Medical
Sciences in Poznan, Poznań, Poland
The presentation aims to describe the early Polish neurology and its pioneer, Professor Jan Pilz. During his medical study in Zurich and Bern,
Jan Pilz was so impressed by Swiss achievements in psychiatry that he
decided to work in psychiatric clinics in Zurich and Geneva. He took
his doctorate in neuropathology under a well-known Russian physician,
Bekhterew. Pilz visited neurological and psychiatric clinics in France,
Germany, The Netherlands, and Russia. From 1905, he took the first
Polish Department of Neurology and Psychiatry in Cracow. Professor
Pilz was engaged in study of the topography of cortical pupilary motor
centers in animals. He took into consideration only isolated pupilary
movements, which earlier investigators as Bekhterew, Schiff, BrownSéquard had not considered. Pilz proved that there were not only isolated pupilary centers in the cortex but that it was also possible to point
out in the cortex of various species of animals an analogy in the topography of the contraction and expansion centers of the pupils. He also
was interested in hereditariness of mental diseases and homosexuality.
Pilz was the President of Eugenics Society in Cracow, a founder of the
Neurological and Psychiatric Society, the Society for Caring Mental
Patients. His co-workers were first researchers on neurological and
psychiatric diseases in Poland. References: Archive of Jagiellonian
University in Cracow, Jan Pilz’s records.
H.07
ROMUALD MINKIEWICZ (1878–1944) – FOUNDER
OF THE WARSAW SCHOOL OF ETHOLOGY
Chmurzyński J., Korczyńska J., Godzińska E.J.
Laboratory of Ethology, Department of Neurophysiology, Nencki
Romuald Minkiewicz was an outstanding Polish biologist and
ethologist, as well as a devoted socialist activist. After biological studies in St. Petersburg (Russia), he acted as assistant at the
University of Kazan, where he received (1904) his PhD degree.
Initially, he was interested in hydrobiology and biological oceanology, and studied in various marine biological stations. After
the WW I, he was active in the fields of physiology of perception,
memory, acquired and innate behaviour of animals, and worked
as professor at the Free Polish University and in the Nencki Institute of Experimental Biology in Warsaw (Head of the Department
of General Biology, 1918–1939). In 1926–1931 he was Chairman
of that Institute. Minkiewicz was interested in taxes (in that time
called “tropisms”), in particular in photic reactions of various animals to colour lights. He was among the first ones to study modifications of body colouring adapting animals to their environment,
including the so called “disguising behaviour” of the crab Maia
squinado and behavioural patterns employed by animals to find
sites matching their own colouration. He also studied ethology of
ants and of flying aculeate Hymenoptera. He devoted an extensive
study to nest architecture and prey of digger wasps (Sphegidae),
today still cited in relevant papers. He wrote poems and dramas.
He died of injury in 1944 during Warsaw uprising.
H.08
STEFAN BOROWIECKI (1881–1937) – THE FOUNDER
OF THE DEPARTMENT OF NEUROLOGY
AT THE POZNAN UNIVERSITY
Magowska A.
Sciences in Poznan, Poznań, Poland
The presentation aims to describe the life and works of Professor Stefan Borowiecki (1881–1937), a typical representative of
the first generation of Polish neurologists, well educated in both
neurology and psychiatry at Swiss, German, Russian and French
universities. As a young doctor, he took a job at the hospital for
mental patients in Kochanówka near Łódź. Next, he rounded out
his medical education at the outpatient’s clinic run by Professor
Jan Pilz in Kraków, the anatomical laboratory run by Prof. K.
Monakow, the hospital wards run by Dr. J. Babiński in Paris, and
some hospitals in Rheinau, Berlin, and Zurich. For some years he
run a ward for nervous and mental diseases at the St. Lazarus’s
Hospital in Kraków. From 1921 to his premature death, he had
taken the Chair of Neurology and Psychiatry at the Poznan University. He carried out research on brain anatomy, congenital brain
defects, psychoanalysis, and mechanism of persecution. References: Public Record Office in Warsaw and Archive of the University of Medical Sciences in Poznań, S. Borowiecki’s records.
H.09
MAKSYMILIAN ROSE (1883–1937) – THE WORLD
FAMOUS POLISH NEUROANATOMIST
Narkiewicz O.1, Lewicki K.2
1
Department of Anatomy and Neurobiology, 2 Department of Surgery
and Urology for Children and Adolescents, Medical University of
Gdańsk, Gdańsk, Poland
Maksymilian Rose was born in 1883. In 1908 he graduated from
faculty of medicine at Jagiellonian University in Cracow. Thereafter he worked in Berlin with T. Ziehen and H. Oppenheim specializing in neurology. Working with K. Brodmann in clinic of
R. Gaupp in Tubingen he became interested in anatomy of the
brain and especially in cytoarchitectonic of the cerebral cortex.
Anatomy was still in the centre of his attention while he was working in Anatomy Institute under direction of K. Kostanecki. Between 1925–1928 he was appointed as director of the Department
of Neurology in the Kaiser Wilhelm Institute in Berlin. Between
1928–1931 he worked as assistant professor (docent) at the Warsaw
University. At the same time, in Warsaw he organized one of the
first Institutes for Brain Research in Europe. He moved to Wilno,
where he was appointed in the University in 1931 as head of the
psychiatric clinic, and since 1934 as head of the neurological clinic
as well. In the same time he moved the Warsaw Brain Research Institute to Wilno and became his Director. The cytoarchitectonic of
the cerebral cortex was his main topic of research. He founded the
theory of the division of the cortex based on its development. He
was the author of several cytoarchitectonic atlases and many scientific publications from neuroanatomy, neurology and psychiatry
field. Professor Rose died in 1937 in Wilno.
H.10
JAN DEMBOWSKI (1889–1963), ANIMAL
PSYCHOLOGIST AND FOUNDER OF BEHAVIOURAL
PROTISTOLOGY IN POLAND
Chmurzyński J.1, Korczyńska J.1, Mirgos A.2, Godzińska E.J.1
1
Laboratory of Ethology, Department of Neurophysiology,
2
Information Technology Unit, Nencki Institute of Experimental
Jan Dembowski was an outstanding Polish biologist, protistologist
and animal psychologist. After biological studies in St. Petersburg
(Russia), he received his PhD degree at the Warsaw University. He
was then professor at the Free Polish University, and worked at the
Nencki Institute of Experimental Biology in Warsaw, and then at
the Stephen Báthory University in Vilnius. After WW II he worked
at the Łódź University, and took part in the restoration of the Nencki
Institute, where he became Head of the Department of Biology and
Director (1947–1960). He was the first President of the new Polish
Academy of Sciences (1952–1956). Dembowski wrote numerous
books and scientific papers, both theoretical and experimental. His
most important studies dealt with the behaviour of a protozoan Paramecium caudatum, larvae of the caddisfly Molanna angustata and
various crabs. In Paramecium he analysed, among others, active
choice of food on the basis of its physico-chemical features, patterns of swimming and geotactic behaviour. He also demonstrated
the inability of Paramecium to show avoidance learning in response
to light or shadow coupled with the electric shock. His research on
behaviour of arthropods was focused mainly on the question of
behavioural plasticity. In his book “Animal Psychology” (1950) he
put forward a precursory thesis of scientific revolutions, broadly accepted only in 1962 when it was proposed again by Th. S. Kuhn.
Dembowski played the piano and was a passionate hunter.
H.11
STEFAN KAZIMIERZ PIEŃKOWSKI (1885–1940)
– THE FORGOTTEN POLISH NEUROLOGIST
AND PSYCHIATRIST
Magowska A.
Sciences in Poznań, Poznań, Poland
This presentation shows a profile of Professor Stefan Kazimierz
Pieńkowski, the eminent neurologist and psychiatrist, the successor
of Professor Jan Pilz at the Department of Neurology and Psychiatry at the Jagiellonian University in Cracow. He studied medicine in
Warsaw and Kiev, next, medicine, philosophy and natural sciences
in Cracow. He specialized in embryology, bacteriology, neurology
and psychiatry in Cracow and some universities in France. His
main scientific interests were focused on hereditariness of mental
diseases, encephalitis, brain injuries, and influence of blood circulation disorder on nervous system. He published papers on encephalitis in French and Polish. He was the Editor-in-Chief of the
journal “Neurologia Polska” and a correspondent for the French
journal “Revue Neurologique”, the Vice-President of the Psychiatric and Neurological Society, the Commission for Research on
the Brain of Marshall Józef Piłsudski, the Eugenic Commission
in the Polish Psychiatric Society. He was murdered by Soviets in
Katyń. References: Public Record Office in Warsaw and Archive
of Jagiellonian University in Cracow, S. K. Pieńkowski’s records.
H. 12
JERZY CHORÓBSKI (1902–1986) – THE FOUNDER
OF POLISH NEUROSURGERY
Platek R., Czarkowska-Bauch J., Skup M.
Jerzy Choróbski, a distinguished neurosurgeon, who established
Polish modern neurosurgery, graduated in medicine from the Jagiellonian University in 1926 and developed his career in European
and American clinics. In 1920s he studied in Paris and mastered
his talent among the elite European neurologists, neurosurgeons
and psychiatrists of those days (Bidziński 2008). In 1930s, as a
grantee of M. Ottman and Rockefeller Foundations, Choróbski
specialized in neurosurgery under supervision of Prof. Penfield
in Montreal. Many of his scientific relations outlasted the time of
World War II and resulted in visits of leading neurosurgeons in Poland. Under difficult facility circumstances, on 6 November 1935
Choróbski opened a 15-bed Neurosurgery Ward at the Department
of Neurological Diseases of the Warsaw University. He considered
that date as the beginning of Polish modern neurosurgery. Choróbski broaden the spectrum of neurosurgeries being performed so far
and many were conducted in Poland for the first time. In addition
to brain, cerebellar or spinal cord tumors, he developed surgical
treatment of sympathetic system diseases, which couldn’t be cured
pharmacologically at that time. Together with progress of diagnostic techniques, Choróbski introduced surgical treatment of epilepsy
based on Penfield school approach and developed therapy of brain
vascular diseases. This talented neurosurgeon established also surgical treatment of involuntary movements (Choróbski 1961, 1962).
H.13
JERZY KONORSKI (1903–1973) – THE PIONEER
OF INVESTIGATIONS ON VOLUNTARY MOTOR
BEHAVIOR
Górska T. 1, Radzikowska Z. 1, Mirgos A. 2
1
Department of Neurophysiology, 2 Information Technology Unit,
Jerzy Konorski was one of the most eminent world leaders in research
on brain physiology, the author of instrumental (Type II) conditioned
reflex theory and the founder of the Polish school of neurophysiology. Already as a student of medicine, Konorski discerned experimentally a new type of conditioned reflexes, which he called motor
(Type II) as opposed to classic (Type I) salivary reflexes, studied by
I.P. Pavlov. In 1928 he described, for the first time, the specificity of
Type II reflexes and the methods of their training. In 1931–1933 he
stayed in Pavlov’s famous laboratory in Petersburg. Since 1933 he
was associated with the Nencki Institute of Experimental Biology,
where after World War II, he created and became the Head of the
376
Department of Neurophysiology, the research of which was devoted
to the analysis of mechanisms controlling voluntary motor behavior.
Konorski published more than 180 papers, among them three monographs, concerning principles of brain functioning. His passion for
research, creativity and profound knowledge attracted many scientists from around the world who visited him and his Department. In
recognition of his scientific achievements Konorski’s name was put
on a strict list of candidates for Nobel Price. His death in 1973 was
a great loss for both the Polish and world scientific community. His
ideas are still appreciated and continue to inspire further research
with the use of modern techniques.
H.14
LILIANA LUBIŃSKA (1904–1990) – THE PIONEER
OF INVESTIGATIONS ON AXONAL TRANSPORT
AND NERVE REGENERATION
Oderfeld-Nowak B.1, Skangiel-Kramska J.1, Górska T.2, Smyda J.2
1
Warszawa, Poland
Professor Liliana Lubińska has been working in the Nencki Institute
since 1933. In 1946 she has founded together with Professor Jerzy
Konorski the Department of Neurophysiology in the Institute. Liliana Lubińska devoted practically her whole life to studies on peripheral nervous system with the main aim to elucidate the mechanism of
functioning of the neuron with its small cell body and long axon. She
has studied thus the events taking place during regeneration and Wallerian degeneration as well as the mechanisms of axonal transport.
She has used simple methods elaborated and tested in every detail.
In the late fifties Professor Liliana Lubińska encouraged Professor
Stella Niemierko – the biochemist, to begin joint studies on transport
of some compounds along axons. The joint work on this project as
well as concomitantly carried on studies with Professor Jirina Zelena, brought to a formulation of basic, largely citated and referred to,
hypothesis of bidirectional movement of axoplasm. Liliana Lubińska
was an outstanding scientist with strong personality and with very
brought knowledge of neurobiological phenomena and a unique intellectual capacity for posing and solving scientific problems. She
has been the author of several original papers and reviews of fundamental significance, still frequently quoted in world literature. They
had a great impact on neurobiology, influenced the research of many
scientists and triggered a new line of experiments.
H.15
STELLA NIEMIERKO (1906–2006) – THE FOUNDER
OF FUNCTIONAL NEUROCHEMISTRY IN POLAND
Oderfeld-Nowak B.1, Skangiel-Kramska J.1, Smyda J.2
1
Department of Molecular and Cellular Neurobiology, 2 Department of Neurophysiology, Nencki Institute of Experimental Biology PAS, Warszawa, Poland
All the long and most fruitful scientific activity of Professor Stella
Niemierko has been inseparably connected with the Nencki Institute of Experimental Biology which she entered in 1926 as a thirdyear student. For many years she was involved in the studies of various aspects of phosphorus metabolism. In 1959 Stella Niemierko
established the first national team to introduce a neurochemical approach to systems based physiology. Four years later she officially
founded the Laboratory of Neurochemistry which she guided until
her retirement in 1977. She actively participated in its further devel-
opment until the end of her life. Collaborative work between Professor Stella Niemierko and Professor. Liliana Lubińska led to significant advances in the field of axonal transport. The major findings
which brought to a formulation of basic hypothesis of bidirectional
movement of axoplasm were published in the leading scientific
journals Nature and Science. Encouraged by Prof. Jerzy Konorski,
she undertook the studies on neurochemical basis of learning and
memory. She also initiated investigations concerning biochemical
changes occurring in the brain after first visual stimulation, which
were awarded a national prize. Professor Niemierko has contributed
to the development of neurochemical research projects in several
centers in Poland gaining the respect and admiration of the whole
Polish biochemical and neurobiological Community. She was honored member of the Polish Neuroscience Society.
H.16
EWA OSETOWSKA (1919–1978)
– DISTINGUISHED POLISH NEUROPATHOLOGIST
Klimkiewicz J., Kaliszek-Kiniorska A., Chabros-Borawska W.
Department of Neuropathology, Mossakowski Medical Research
Professor Ewa Osetowska was one of the most distinguished Polish
neuropathologists. She was born in 1919. In 1958 she underwent training in Bunge Institute, Antwerp, working under the supervision of
Prof. Ludo van Bogaert. This experience together with the inspiration
of her Polish mentor, Prof. Adam Opalski, proved decisive for her following scientific career. In 1963 she became the head of the Neuropathology Department of the Polish Academy of Sciences. She put her
effort into organizing work in this new and completely unknown in
Poland field of science. She assembled a group of people interested in
the field of neuropathology and inspired them to the scientific work and
to the organization of their own laboratories in different towns of the
country. Professor E. Osetowska promoted 20 PhD’s and 5 postdoctoral degrees. Among her students we can find many of later prominent
scientists, such as: Professor Mirosław J. Mossakowski–President of
the Polish Academy of Sciences, Professor Henryk Wiśniewski – Director of the Institute for Basic Research in New York, Professor Maria
Dambska, a renowned specialist in the field of the development of the
nervous system. At the age of 49, after long and severe illness, Professor. Ewa Osetowska passes away, leaving behind a stunning scientific
legacy – altogether over 150 scientific publications in which clinical
and neuropathological diagnosis was always a starting point for further search of the pathogenesis of the diseases.
H.17
MIROSŁAW J. MOSSAKOWSKI (1929–2001)
AND HIS CONTRIBUTION TO NEUROPATHOLOGICAL
RESEARCH IN POLAND
Wójcik L.
Professor Mirosław Mossakowski, was a leading figure in neuropathology and neuroscience in Poland. For more than three decades
he remained active in the study of various neurological disorders.
Foremost was his brilliant early work on the morphological changes
of neural cells exposed to ammonia, copper and sera from patients
with hepatic encephalopathy, highlighting the critical role of astrocytes in the mechanism of this disorder. The biology and pathology
of glial cells studied from the morphological and biochemical points
of view, the observation of primary fibrillary gliosis, were particularly innovative. His studies of neurodegenerative diseases brought
the opportunity to observe and published first the description of the
coexistence of lipid storage diseases and leucodystrophies. His work
on cerebral ischemia brought about one of the first demonstrations of
imbalance between glycogen synthesis and metabolism in the CNS.
In addition to his considerable scientific contributions, he extended
his inexhaustible energy and untiring devotion to the promotion of
neuroscience and neuropathology. Professor Mossakowski served
for two terms as a vice-president of the International Society of
Neuropathology and on the editorial boards of many journals. He
received many prestigious awards and nominations. Professor Mossakowski was a kind and generous man, a superb teacher, and role
model. He will be sorely missed by all who were fortunate to experience his matchless wit, knowledge and wisdom.
H.18
KAZIMIERZ JAN ZIELINSKI (1929–2004) AND HIS
STUDIES ON DEFENSIVE CONDITIONING
Werka T.1, Stasiak M.2, Knapska E.1, Wesierska M.1, Sadowska J.1
1
Biology PAS, Warszawa, Poland; 2 Laboratory of Alimentary
Behavior, National Food and Nutrition
Institute, Warszawa, Poland
Professor Kazimierz Jan Zielinski had his over-forty-year-long career path very closely connected with the development of the Nencki
Institute. The general scope of his research was behavioral neuroscience. Much of his scientific work was devoted to systematic study of
the defensive Pavlovian and instrumental conditioning, and to functions of the prefrontal cortex and the amygdala in aversive learning. Topics, to which Professor Zielinski had made important contributions, ranged from stimulus intensity dynamism to conditioned
inhibition and processes of inhibition of delay. The director of the
Nencki Institute for many terms, he also organized the Laboratory
of Defensive Conditioned Reflexes within the Department of Neurophysiology. The scope of the Laboratory reflects one of Professor
Zielinski’s biggest scientific passions – the quest for understanding
the mechanisms of avoidance learning, as well as internal complexity of this instrumental defensive response. Numerous studies at the
Laboratory, many designed, performed or supervised by himself,
were devoted to study parameters affecting the performance and
differentiation of the avoidance response, and to examine the neuronal processes that mediate the states of fear and safety. Professor
Zielinski hypothesized that different physiological mechanisms are
responsible for performance of short- and long-latency avoidance
responses, and that these two classes of avoidance response evoke
diverse emotional states.
H.19
BOGUSŁAW ŻERNICKI (1931–2002) – INVESTIGATIONS
ON SLEEP, LEARNING AND PLASTICITY
Burnat K., Turlejski K.
Professor Żernicki described himself as a “physician by education, physiologist by profession, but admirer of psychology and
philosophy”. His most known and cited research concerned the
pretrigeminal preparation. He proved that brain, isolated from the
majority of sensory stimuli, preserves its basic behavioral functions and continues to learn. He studied also the perceptive and
associative mechanisms of learning after deprivation of pattern vision at early developmental stages, elaborating the role of subcortical visual pathways in visual development. Professor Żernicki
conducted research at the University of Pisa, University of Paris,
Chilean University in Santiago, University of Rochester, and the
University of Nice. He supervised NIMH grants for the Nencki
Institute, hosted many foreign scientists, and organized several international conferences and meetings. Professor Żernicki was an
author of more than 100 papers on central mechanisms of conditioning, isolated brain, physiology of the visual system and developmental neurophysiology. He also wrote four books in the Recent
Discoveries of Science series, many chapters, textbooks and articles on policy in science. As a mentor in neuroscience he supervised 11 PhD theses. Most of his pupils continue research work in
tenured positions in and outside the Institute. Several generations
of neurophysiologists from the Nencki Institute were inspired by
his scientific passion, talent for research and organization.
H.20
STANISŁAW WOLFARTH (1933–2007) AND HIS WORK
RELATED TO PARKINSON’S DISEASE
Zieba B.1, Ossowska K.2
1
Department of Neurobiology, 2 Department of Neuro- and
Psychopharmacology, Institute of Pharmacology PAS, Kraków,
Poland
Stanisław Wolfarth graduated at the Medical Academy in Kraków
in 1963. He worked for over 40 years at the Institute of Pharmacology PAS, where he was a head of the Department of Neuropharmacology (1971–2003). Studies on the role of the basal ganglia in development of Parkinson’s disease became a passion of his life. One
of his first achievements was the finding that clinical relationship
termed dopaminergic-cholinergic balance resulted from interactions of complex neuronal systems including the substantia nigra,
striatum and other structures. He demonstrated that GABAergic
pathways connecting the substantia nigra, thalamus and zona incerta - lateral hypothalamus controlled the muscle tone, and played
a crucial role in initiation of movements. He was one of the authors
of a prototypic device for objective measurement of the muscle
tone of rat’s hind paw which is now the only apparatus of this type
worldwide. Within the last 15 years he found antiparkinsonian
properties of ligands of NMDA and metabotropic glutamate receptors in animal models, and discovered that muscle stiffness in
the elderly had completely different pathophysiological basis than
parkinsonian muscle rigidity. He was deeply involved in several
international co-operations, was an author of over 90 scientific papers, and for his pioneer discoveries was highly prized by many
scientific organizations. His works are characterized by amazing
coherence of research objectives, consistency and up-to-dateness.

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